IN VIVO GENE THERAPY USING INTRAOSSEOUS DELIVERY OF A LENTIVIRALGENE CONSTRUCT
Methods, compositions, and systems for treating subject(s) in need of plasma Factor VIII, particularly a subject having preexisting anti-FVIII inhibitory antibodies, are provided. The methods involve administering to the subject a therapeutically effective amount of an inflammation suppressor, a therapeutically effective amount of a CD8+ T cell depleting agent, and a therapeutically effective amount of a composition comprising a lentiviral vector (LV) comprising an optimized FVIII expression cassette expressibly linked to a megakaryocyte-specific promoter. Such methods, compositions, and systems are useful to treat subjects with blood clotting disorder(s), such as hemophilia A.
This application claims priority to U.S. Provisional Application No. 62/663,930 filed on Apr. 27, 2018, which is incorporated herein by reference in its entirety as if fully set forth herein.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with government support under Grant Nos. HL123326-01A1 and HL134621-01, awarded by the NIH/NHLBI. The government has certain rights in the invention.
FIELD OF THE DISCLOSUREThe present disclosure provides compositions and methods for in vivo gene therapy, for instance using intraosseous delivery or other delivery modes. Examples of such therapy provide platelet-specific expression of activity-enhanced plasma Factor VIII, for instance for treatment of hemophilia and other blood clotting disorders.
BACKGROUND OF THE DISCLOSUREInhibitory antibody formation against human factor VIII (FVIII) is a significant complication in treatment of hemophilia A (HemA) patients. Ectopic expression of FVIII in platelets could be an effective approach for treating HemA. Unlike circulating plasma FVIII, FVIII stored in α-granules of platelets is protected from being processed by antigen presenting cells, therefore significantly reducing the chance to induce anti-FVIII immune responses. Moreover, platelet FVIII is not neutralized by preexisting anti-FVIII inhibitory antibodies (inhibitors), which will benefit HemA patients who already developed inhibitors. During bleeding, the activated platelets locally excrete FVIII that can directly participate in the coagulation cascade and promote clot formation. It has recently been demonstrated that low levels of platelet FVIII can partially correct hemophilia phenotype in animal models.
One approach to direct FVIII long-term expression in platelets is intraosseous (IO) infusion of lentiviral vectors (LVs) carrying a FVIII transgene controlled by a megakaryocyte specific promoter Gp1bα (Gp1bα-F8-LVs). In this in vivo gene therapy protocol of HemA, hematopoietic stem cells (HSCs) was efficiently transduced by LVs in situ, followed by FVIII transgene expression in the late stage of megakaryocyte differentiation, and finally storage of FVIII protein in α-granules of platelets. Previous work showed that a single IO infusion of Gp1bα-F8-LV resulted in over 3% platelets containing hFVIII, leading to partial phenotype correction in immune competent HemA mice with and without pre-existing inhibitors.
Furthermore, in vivo delivery of LVs can avoid many difficulties and potential toxicities encountered by ex vivo gene therapy including loss of stem cell properties and engraftment potential after cell transfer, negative effects of ex vivo cytokine stimulation and pre-conditioning of the subject. However, one potential limitation of in vivo gene therapy protocols is the induction of LV-specific immune responses, which may decrease LV transduction efficiency and eliminate LV transduced cells.
HemA, characterized by a deficiency of functional plasma FVIII, is an ideal disease candidate for gene therapy to attain long-term therapeutic FVIII levels. An optimized transgene for effective in vivo gene therapy of hemophilia would confer a higher gene expression level and a transgene product with enhanced biological activity. Since the size of a full-length human FVIII cDNA is quite large (>7 kbp), it cannot be easily packaged in viral vectors, often resulting in low titers of viruses. Shorter cDNAs coding for FVIII variants have therefore been used in gene therapy preclinical research. For instance, a B-domain deleted FVIII (BDD-FVIII) variant exhibits similar functional activity as the full-length FVIII. Additional mutations have been incorporated into the gene coding for the BDD-FVIII to increase its secretion, such as, F8/N6 with a 226 aa B-domain variant sequence (Miao et al., Blood 103:3412-3419, 2004), codon optimized BDD-FVIII and F8/N6 (Ward et al., Blood 117:798-807, 2011), F8-V3 (McIntosh et al., Blood 121:3335-3344, 2013) with a 17-aa peptide coding sequence replacing the 226 aa/N6 in F8/N6, and F8-RH encoding a BDD-FVIII variant with R1645H (Siner et al., Blood 121:4396-4403, 2013). Some FVIII variants, like FVIII-RH and furin-cleavage site deleted BDD-FVIII variants (Nguyen et al., J Thromb Haemostas. 15:110-121, 2017), exhibit an increase in biological activity compared with BDD-FVIII, likely due to its slower dissociation of the A2-domain upon thrombin activation.
U.S. Pat. No. 5,779,708 describes an intraosseous and/or hard tissue drug delivery device and method that perforates hard tissue and provides a drug delivery passage into underlying tissue. The device includes a hollow drill bit and a stylet removably inserted into the hollow drill bit. The stylet, which keeps the bore of the hollow drill bit from plugging up during drilling, is removed after drilling to permit the operator to inject medication through the bore of the hollow drill bit.
In spite of previous work, there still exists a need for safe and reliable methods of replacing or supplementing functional FVIII in a subject. There is a particular need for methods which do not rely on pre-conditioning or myeloablative treatments, and which can provide therapeutic benefit (such as appropriate clotting function) in spite of the presence of inhibitory anti-FVIII antibodies in the subject to be treated.
SUMMARY OF THE DISCLOSUREDescribed herein is the development of clinically relevant strategies for treating HemA. In examples of these strategies, self-inactivating lentiviral vectors (LV) carrying various different FVIII variant transgenes (including variants optimized for expression in this system) driven by a platelet-specific promoter Gp1bα (G) were delivered into HemA subjects without preconditioning. In some embodiments, this transgene is delivered via intraosseous (IO) administration (for instance into the tibia or iliac bone). In other embedment's, delivery is intranasal (or otherwise to the lung) or intravenous. The provided FVIII transgenes can effectively transduce hematopoietic stem cells (HSCs), largely without elicitation of anti-FVIII inhibitory antibodies (anti-FVIII inhibitors). The resulting FVIII is expressed and stored in platelet α-granules, and can at least partially correct bleeding phenotype in immune-competent HemA subjects, even in the presence of pre-existing anti-FVIII inhibitors.
Embodiments of the current disclosure describe compositions and methods that enable combination treatments including IO delivery of LVs expressing a platelet-targeted modified FVIII gene. for treatment of hemophilia. The use of IO delivery for efficient transduction of HSCs in vivo results in a low inflammatory and/or immune response in the subject, accompanied by low systemic toxicity. This is particularly beneficial for certain subjects, including for instance subjects with a bleeding disorder such as hemophilia.
Additional embodiments provide a method of treating a subject in need of plasma Factor VIII, including: administering to the subject a therapeutically effective amount of an inflammation suppressor (such as dexamethasone (Dex)); administering to the subject a therapeutically effective amount of a CD8+ T cell depleting agent; and administering to the subject a therapeutically effective amount of a composition including a lentiviral vector including an optimized FVIII expression cassette expressibly linked to a megakaryocyte-specific promoter, wherein administration is via intraosseous (IO) infusion; intravenous (IV) delivery; or intranasal (IN) or inhaled delivery.
In examples of this method of treatment embodiment, the dexamethasone is administered in doses of 100 mg/kg at −24 h before, −4 h before, 4 h after, and 24 h after administration of the LV.
In examples of the provided embodiments, the CD8+ T cell depleting agent includes an anti-CD8 antibody. For instance, in examples, the CD8+ T cell depleting agent is an anti-CD8α mAb administered in doses of 4 mg/kg at −1 day before, 4 days after, and 11 days after delivery of the LV.
In other examples of the provided embodiments, the megakaryocyte-specific promoter is a GP1b-alpha promoter.
Specifically contemplated in examples of the provided embodiments are methods that do not include pre-conditioning or myeloablative treatment of the subject.
In more examples of the provided embodiments, the IO infusion is carried out at a rate of 2 μL/min to 15 μL/min, 5 μL/min to 12 μL/min, or at no more than 10 μL/min. For instance, the IO infusion in some examples is carried out at a rate of 0.01 mL/min to 0.5 mL/min, 0.05 mL/min to 0.3 mL/min, 0.1 mL/min to 0.25 mL/min, or at no more than 0.4 mL/min. Optionally, the IO infusion is carried out over a period of no more than 45 minutes.
In yet additional examples, the optimized FVIII expression cassette includes hF8X10 K12 (SEQ ID NO: 1) or hF8/N6K12RH (SEQ ID NO: 2). In any of the provided embodiments, the lentiviral vector may include SEQ ID NO: 3 or SEQ ID NO: 4, or a functional variant thereof.
Also provided are examples of the herein provided methods, which are methods for treating a hemostasis related disorder in the subject, and the subject is in need of such treatment. By way of example, the subject may have hemophilia A (hemA), von Willebrand disease, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated vascular coagulation (DIC), or over-anticoagulation treatment disorder.
In yet another embodiment of the provided treatment method, the subject is a HemA subject with preexisting anti-FVIII inhibitory antibodies.
Also provided are methods of treating a subject in need of plasma Factor VIII, essentially as described herein.
At least one of the drawings submitted herewith is better understood in color. Applicant considers the color versions of the drawing(s) as part of the original submission and reserve the right to present color images of the drawings in later proceedings.
The disclosed nucleic and/or amino acid sequences are shown using standard letter abbreviations for nucleotide bases, and one or three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. A computer readable text file, entitled “Sequence Listing.txt” created on or about Apr. 26, 2019, with a file size of 48 KB, contains the sequence listing for this application and is hereby incorporated by reference in its entirety. In the accompanying Sequence Listing and/or as provided herein:
SEQ ID NO: 1 is the nucleic acid sequence of the insert BDDFVII1X10K12, which encodes the human FVIII variant F8X10K12; it differs from variant F8/N6 by a deleted B-domain, a 10-amino acid change in the A1 domain and a 12-amino acid change in the light chain.
SEQ ID NO: 2 is the nucleic acid sequence of the insert BDDFVIIIn6K12RH, which encodes the human FVIII variant F8/N6K12RH; it differs from variant F8/N6 at a 12-amino acid change in the light chain and an amino acid change at the furin cleavage site within the B domain (position R1645H).
SEQ ID NO: 3 is the nucleic acid sequence of pRRL-GP1balpha-hF8X10K12-WPRE; the F8 cDNA insert hF8X10K12 (SEQ ID NO: 1) is at positions 2320-6738 of this sequence, which also includes the lentiviral vector backbone, platelet-specific GP1b alpha promoter, and WPRE element inserted in the vector to make stable mRNA.
SEQ ID NO: 4 is the nucleic acid sequence of pRRL-GP1balpha-hF8/N6K12RH-WPRE; the F8 cDNA insert hF8/N6K12RH (SEQ ID NO: 2) is at positions 2308-7414 of this sequence which also includes the lentiviral vector backbone, platelet-specific GP1b alpha promoter, and WPRE element inserted in the vector to make stable mRNA.
SEQ ID NOs: 5 and 6 are, respectively, the nucleotide sequences of forward and reverse amplification primer for GAG.
SEQ ID NO: 7 is the nucleotide sequence of a probe for GAG.
SEQ ID NOs: 8 and 9 are, respectively, the nucleotide sequences of forward and reverse amplification primer for human Factor VIII.
SEQ ID NOs: 10 and 11 are, respectively, the nucleotide sequences of forward and reverse amplification primer for Rpl19.
DETAILED DESCRIPTIONHemophilia A (HemA) is a X-linked inherited or acquired genetic disease with defective plasma factor VIII (FVIII), resulting in unstable blood clot formation in HemA patients when internal and/or external bleeding occurs. Described herein are gene therapy protocols to treat hemophilia A and other bleeding disorders by direct infusion of lentiviral vectors (LVs) (or other expression units) carrying an engineered FVIII gene into bone marrow to efficiently transduce hematopoietic stem cells (HSCs).
In example embodiments, FVIII expression is targeted specifically to platelets using a megakaryocyte-specific promoter to drive transgene expression; this results in FVIII-contain platelets circulating in the blood. Expression the platelets prevents or significantly inhibits FVIII secretion into blood, and thus prevents an anti-FVIII immune response in the subjects so treated. Immune response to FVIII disadvantageously leads to neutralization of this factor by anti-FVIII antibodies (so called anti-FVIII inhibitors) in hemophilia patients. Expression inside of platelets maintains the FVIII inside the platelets until they are triggered by the clotting cascade, leading to release of FVIII when bleeding occurs.
Based on results reported here, it is believed that a single gene therapy treatment via in vivo delivery of G-F8-LVs driven by a megakaryocyte-specific promoter directly into bone marrow will benefit HemA patients, especially patents with anti-FVIII inhibitors.
Beneficially, and in contrast to traditional ex vivo gene therapy, the IO in vivo delivery system provided herein avoids the requirement for both ex vivo cell manipulation as well as use of fully or partially myeloablative condition regimens. Such pre-conditioning is usually required for successful ex vivo gene therapy.
In embodiments described herein, it can be beneficial to precisely control the speed (rate) of IO infusion, as this influences the efficiency at which the IO delivered LVs are transduced into cells in the bone marrow (such as HSCs). Low delivery speeds can be used, and may be beneficial in smaller subjects. Relatively low speeds include, but are not limited to 2 μL/min to 15 μL/min, 5 μL/min to 12 μL/min, or at no more than 10 μL/min. One particular relatively low speed is 0.01 mL/min. A relatively higher delivery speed (rate) can be used in larger animals, including humans. By way of example, a relatively higher rate of delivery is carried out at 0.01 mL/min to 0.5 mL/min, 0.05 mL/min to 0.3 mL/min, 0.1 mL/min to 0.25 mL/min, or at no more than 0.4 mL/min. Clearly, a faster rate of delivery will shorten the infusion time, and is therefore considered beneficial so long as it is selected to be such a rate that will still achieve efficient transduction in the specific subject being treated. By way of example, he total volume of the vector bolus used to treat a relatively large subject (such as a dog) may be 2-20 mL or more, and it may be beneficial to the subject to complete infusion in 20-40 mins.
Compared to existing, proposed treatments for hemophilia A or B that rely on recombinant adeno-associated viral (AAV) vectors (rAAV), lentiviral gene therapy as described herein has several advantages. First, AAV persists as episomal, concatemerized vector following in vivo gene transfer. Over time in that situation, transgene expression may decrease and it is expected that repeated dosing will be required. It remains to be shown whether such repeated dosing is feasible, or effect. In contrast, lentiviral vectors are an integrative vector that is incorporated into the genome of the transduced cells. If successfully integrated into long-lived bone marrow HSCs, as taught here, a single treatment of LV-mediated gene therapy may be sufficient for life-long therapeutic benefit. In addition, a significant proportion of human subjects have pre-existing neutralizing antibodies to various AAV serotypes, and nearly all subjects will develop antibodies to the serotype utilized for initial treatment. This will make repeated dosing with the same serotype challenging if not impossible.
AAV is also a poor choice for expression of FVIII cDNA because its transgene capacity is simply too small. Further, LV can efficiently transduce both dividing and no-dividing cells, leading to significant increases in efficiency in targeting primitive HSCs, compared to AAV and traditional gamma-retroviral vectors which can only transduce dividing cells.
Furthermore, recent clinical trials of ex vivo lentiviral gene therapy for several primary deficiency diseases showed significant clinical benefit without adverse effects or aberrant clonal expansion.
Compared to other in vivo LV-mediated gene therapy approaches, IO delivery is unique. Among other benefits (including benefits specifically described elsewhere herein), slow injection of LVs into the bone marrow cavity can avoid (substantially or completely) the inflammation and systemic toxicity caused by systemic intravenous (IV) deliver of AAV and other candidate gene therapy vectors.
In certain embodiments, it can be beneficial to use one or more compounds to facilitate or enhance efficient transduction of the target HSCs. For example, several compounds (such as dexamethasone, anti-CD8 monoclonal antibodies, and staurosporine) can enhance LV transduction of HSCs. Another such compound is rapamycin, and therefore example methods involve the co-administration, for instance via IO, of LV and rapamycin to enhance the rate of transduction. This can be particularly beneficial in instances where the viral titer may be low or below a desired level. Another method to facilitate or enhance LV transduction efficiency is to conjugate the LVs with ligand(s) or antibody fragment(s) that can specially recognize receptors on HSCs, thus targeting the LV to the preferred cell for transduction.
Additional options and embodiments of the disclosure are now described in more detail.
Any composition formulation disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration, whether for research, prophylactic and/or therapeutic treatments. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety and purity standards as required by United States FDA Office of Biological Standards and/or other relevant foreign regulatory agencies.
Exemplary generally used pharmaceutically acceptable carriers include any and all bulking agents or fillers, solvents or co-solvents, dispersion media, coatings, surfactants, antioxidants (e.g., ascorbic acid, methionine, vitamin E), preservatives, isotonic agents, absorption delaying agents, salts, stabilizers, buffering agents, chelating agents (e.g., EDTA), gels, binders, disintegration agents, and/or lubricants.
Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers and/or trimethylamine salts.
Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.
Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.
Exemplary stabilizers include organic sugars, polyhydric sugar alcohols, polyethylene glycol; sulfur-containing reducing agents, amino acids, low molecular weight polypeptides, proteins, immunoglobulins, hydrophilic polymers, or polysaccharides.
Intranasal administration is a non-invasive route for gene therapy delivery that offers advantages such as ease of administration, rapid onset of action, and avoidance of first pass metabolism, gastric acid and enzymatic degradation (Grassin-Delyle et al, Pharmacol Ther. 134:366-379, 2012). Lentiviral vectors can readily be administered intranasally. The GP64-FIV lentiviral vector has been successfully delivered through repeated intranasal administration without significant decline in transgene expression (Podolska et al., Adv Clin Exp Med. 21:525-34, 2012). Methods of intranasal delivery include nasal deposition or instillation, direct instillation into the trachea or lower airways using a catheter or bronchoscope, or aerosolization and inhalation (Weiss, Molecular Therapy 6:148-152, 2002).
In some embodiments, the lentiviral vector is administered intranasally via nasal deposition, a process wherein the site of deposition may influence the route of absorption and target organ distribution. For example, absorption at the mucosa beyond the nasal valve is more likely to happen through the carotid artery providing a direct route to the brain (Djupesland, Drug Deliv Transl Res. 3:42-62, 2013).
In other embodiments, the lentiviral vector is delivered through direct instillation into the trachea or lower airways, for instance using a catheter or bronchoscope. Direct vector instillation can be performed though bronchoscopy administration resulting in heterogenous deposition in parts of the lung, and the use of spray devices directed through the bronchoscope can improve vector distribution and deposition (Weiss, Molecular Therapy 6:148-152, 2002).
In some embodiments the lentiviral vector is delivered through aerosolization and inhalation, a clinically convenient approach that can result in a diffused transgene distribution in the lungs. During aerosolization and inhalation, different nebulization equipment and methods can be used to produce homogenously sized aerosol droplet particles that are deposited in bronchiolar and distal airways (Weiss, Molecular Therapy 6:148-152, 2002).
In some embodiments, the lentiviral vector is delivered as an aerosol spray from a pressure container or dispenser which contains a suitable propellant (such as carbon dioxide), or a nebulizer (e.g., US 2005/0251872), or by other methods including vapor inhalers, Rhinyle catheters, multi-dose droppers, unit-dose pipettes, squeeze bottles, multi-dose metered-dose spray pumps, single/duo-dose spray pumps, slow spray HFA pMDI's, pulsation membrane nebulizers, powder spray devices and nasal inhalers and insufflators (Djupesland, Drug Deliv Transl Res. 3:42-62, 2013).
Lentiviral vectors that carry transgenes also can be delivered intravenously to treat human diseases. See, for instance, US 2000/70190030, US 2005/0251872, CA 2,296,319, US 2007/0190030; Carbonaro et al., Mol Ther. 13:1110-1120, 2006. Intravenous (IV) administration refers to the process of administering a compound directly into a patient's or animal's vein using a needle or tube. Intravenous administration is used wherein a rapid onset of action is required or when other delivery routes are not available due to characteristics of the compound or patient factors (Jin et al., Patient Prefer Adherence. 9:923-942, 2015). Thus, upon injection, the compound or composition can be rapidly delivered to organs bypassing absorptions barriers, but with the potential to elicit toxic effects (Maddison et al., Small Animal Clinical Pharmacology, Second Edition, Chapter 2, 27-40, 2008).
Intravenous administration offers several advantages, including minimal delay for drug availability, the possibility to achieve constant plasma concentrations at a required level, the avoidance of unexpected side effects by stopping the infusion, an alternative route when compounds are poorly absorbed by the gastrointestinal tract, and the avoidance of pain when other administration routes are painful (Claassen, Neglected factors in pharmacology and neuroscience research, Chapter 2, pp 5-22; Huston Ed., 1994).
Most intravenous injections are performed in one of the superficial veins normally used to collect blood, but the choice of vein for intravenous injection depends on several factors (well known to those of skill in the art), including ease of venipuncture, age, whether anesthesia or chronic catheterization is required, and type of composition injected. In some embodiments, the injection of the composition containing a lentiviral vector is made into the jugular, caudal, femoral, lateral marginal, dorsal metatarsal, saphenous, lingual, or dorsal penile vein.
In some embodiments, the technique used to deliver a lentiviral vector can include a clean technique using a sterile syringe and needle, a strict aseptic technique for chronic catheterization, and distal punctures in a peripheral vein when multiple injections are required. In some embodiments, the compound or composition can be delivered by rapid injection into the vein using a syringe, administered intermittently over a specific amount of time using an IV secondary line, or continuously mixed in a main IV solution.
In other embodiments, a lentiviral vector is delivered using one of various different types of catheters, including peripheral IV (PIV), an intravenous catheter inserted by percutaneous venipuncture into a peripheral vein for short-term IV therapy, or a central venous catheter (CVC) that is inserted into a large vein in the central circulation system. In some embodiments, the catheterization method used to deliver the lentiviral vector includes IV infusion, pump infusion, drip infusion, a tunneled catheter, an implanted port, and a peripherally inserted central catheter.
Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions, or dispersions and/or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In some embodiments, suitable carriers for intravenous administration of a lentiviral vector include physiological saline, bacteriostatic water, Cremophor® ELTM (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS).
In all cases, the composition should be sterile and should be fluid to the extent that it is easy to inject. In various embodiments, the pharmaceutical formulation is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. In general, the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (including glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maintained by using a coating, such as lecithin, keeping the required particle size (in the case of dispersions), and using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, including parabens, chlorobutanol, phenol, ascorbic acid and thimerosal. In some embodiments, isotonic agents, including sugars and polyalcohols, such as mannitol, sorbitol and sodium chloride, can be added to the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, such as aluminum monostearate and gelatin (US 2005/0251872).
Sterile injectable solutions can be prepared by incorporating the active compound (such as a lentiviral vector, or another expression vector) in the required amount in an appropriate solvent with one, or a combination, of the ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof (US 2005/0251872).
Unless otherwise indicated, the practice of the present disclosure can employ conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA. These methods are described in the following publications. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); F. M. Ausubel, et al. eds., Current Protocols in Molecular Biology (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., eds. PCR 2: Practical Approach (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual (1988); and R. I. Freshney, ed. Animal Cell Culture (1987).
Sequence information provided by public database can be used to identify gene sequences to target and nucleic acid sequences encoding phenotype-altering proteins as disclosed herein. Exemplary sequences are provided herein.
Variants of the sequences disclosed and referenced herein are also included. Variants of proteins can include those having one or more conservative amino acid substitutions. As used herein, a “conservative substitution” involves a substitution found in one of the following conservative substitutions groups: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser), Threonine (Thr); Group 2: Aspartic acid (Asp), Glutamic acid (Glu); Group 3: Asparagine (Asn), Glutamine (Gin); Group 4: Arginine (Arg), Lysine (Lys), Histidine (His); Group 5: Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val); and Group 6: Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp).
Additionally, amino acids can be grouped into conservative substitution groups by similar function or chemical structure or composition (e.g., acidic, basic, aliphatic, aromatic, sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile. Other groups containing amino acids that are considered conservative substitutions for one another include: sulfur-containing: Met and Cysteine (Cys); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information is found in Creighton (1984) Proteins, W.H. Freeman and Company.
Variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and/or mutations that do not affect the function of an encoded product to a statistically-significant degree.
Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.
“% sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences. “Identity” (often referred to as “similarity”) can be readily calculated by known methods, including (but not limited to) those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wis.). Multiple alignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wis.); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y. Within the context of this disclosure it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. As used herein “default values” will mean any set of values or parameters, which originally load with the software when first initialized.
EXEMPLARY EMBODIMENTSA first embodiment provides a method of treating a subject in need of plasma Factor VIII, the method involving administering to the subject a therapeutically effective amount of an inflammation suppressor; administering to the subject a therapeutically effective amount of a CD8+ T cell depleting agent; and administering to the subject via intraosseous (IO) infusion a therapeutically effective amount of a composition including a lentiviral vector (LV) including an optimized FVIII expression cassette expressibly linked to a megakaryocyte-specific promoter. It is specifically contemplated that administration of the inflammation suppressor and/or the CD8+ T cell depleting agent may occur before, after, or concurrently with administration of the IO infusion, and may occur at the same or different times.
Notably, examples of such treatment methods do not involve pre-conditioning or myeloablative treatment of the subject.
The inflammation suppressor in examples of such methods is dexamethasone (Dex). By way of nonlimiting example, the inflammation suppressor (such as dexamethasone) is administered in doses of 100 mg/kg at −24 h before, −4 h before, 4 h after, and 24 h after IO infusion of the LV.
The CD8+ T cell depleting agent in examples of the provided methods includes an anti-CD8 antibody, such as an anti-CD8α mAb. For instance, in some cases the anti-CD8α mAb is administered in doses of 4 mg/kg at −1 day before, 4 days after, and 11 days after IO infusion of the LV.
In any of the provided methods, the megakaryocyte-specific promoter may be a GP1b-alpha promoter.
Generally, the IO infusion in provided treatment methods is carried out under a controlled, relatively slow rate in order to facilitate effective transduction by the provided LV of cells in the bone marrow. In specific example methods, the IO infusion is carried out at a rate of 2 μL/min to 15 μL/min, or 5 μL/min to 12 μL/min, or at no more than 10 μL/min. In additional examples, for instance when a relatively large (more than 2 kilograms) subject is being treated, the IO infusion is carried out at a rate of 0.01 mL/min to 0.5 mL/min, 0.05 mL/min to 0.3 mL/min, 0.1 mL/min to 0.25 mL/min, or at no more than 0.4 mL/min. Optimally in some embodiments, IO infusion is carried out over a period of no more than 45 minutes.
Beneficially, the transgenic FVIII expressed from a LV delivered intraosseously using a method provided herein is expressed in platelets at a therapeutically relevant (that is, relatively high) level, and is functional in the subject being treated upon clotting cascade triggering of the expressing platelets. By way of example, the optimized FVIII expression cassette includes the hFVIII variant hF8X10 K12 (SEQ ID NO: 1) or the hFVIII variant hF8/N6K12RH (SEQ ID NO: 2). Though in no way intended to be limiting, the lentiviral vector used in such methods can include SEQ ID NO: 3 or SEQ ID NO: 4 (respectively), or a functional variant thereof which is capable of expressing the variant factor VIII in platelets of the subject.
In any of the provided embodiments, the method of treating a subject in need of plasma Factor VIII may be a method for treating a hemostasis related disorder in the subject, specifically wherein the subject is in need of such treatment. By way of example, the subject receiving treatment with methods described herein include subjects that have hemophilia A (hemA), hemophilia B (hemB), von Willebrand disease, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated vascular coagulation (DIC), or over-anticoagulation treatment disorder.
Specifically encompassed herein are treatment methods where the subject is a HemA subject with preexisting anti-FVIII inhibitory antibodies. The platelet-limited hFVIII expression offered by Applicant's methods are particularly beneficial to such subjects, as the expressed FVIII variant is not exposed and circulating in the blood and therefore does not elicit an immune response from circulating anti-FVIII inhibitory antibodies.
The Exemplary Embodiments and Examples below are included to illustrate particular embodiments of the disclosure. Those of ordinary skill in the art will recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain like or similar results without departing from the spirit and scope of the disclosure.
EXEMPLARY EMBODIMENTS1. A method of treating a subject in need of plasma Factor VIII, including: administering to the subject a therapeutically effective amount of an inflammation suppressor; administering to the subject a therapeutically effective amount of a CD8+ T cell depleting agent; and administering to the subject a therapeutically effective amount of a composition including a lentiviral vector including an optimized FVIII expression cassette expressibly linked to a megakaryocyte-specific promoter, wherein administration is via intraosseous (IO) infusion; intravenous (IV) delivery; or intranasal (IN) or inhaled delivery.
2. The method of embodiment 1, wherein the inflammation suppressor includes dexamethasone (Dex).
3. The method of embodiment 2, wherein the dexamethasone is administered in doses of 100 mg/kg at −24 h before, −4 h before, 4 h after, and 24 h after administration of the LV.
4. The method of embodiment 1, wherein the CD8+ T cell depleting agent includes an anti-CD8 antibody.
5. The method of embodiment 4, wherein the CD8+ T cell depleting agent is an anti-CD8α mAb administered in doses of 4 mg/kg at −1 day before, 4 days after, and 11 days after delivery of the LV.
6. The method of embodiment 1, wherein the megakaryocyte-specific promoter is a GP1b-alpha promoter.
7. The method of embodiment 1, which method does not include pre-conditioning or myeloablative treatment of the subject.
8. The method of embodiment 1, wherein the IO infusion is carried out at a rate of 2 μL/min to 15 μL/min, 5 μL/min to 12 μL/min, or at no more than 10 μL/min.
9. The method of embodiment 1, wherein the IO infusion is carried out at a rate of 0.01 mL/min to 0.5 mL/min, 0.05 mL/min to 0.3 mL/min, 0.1 mL/min to 0.25 mL/min, or at no more than 0.4 mL/min.
10. The method of embodiment 1, wherein the IO infusion is carried out over a period of no more than 45 minutes.
11. The method of embodiment 1, wherein the optimized FVIII expression cassette includes hF8X10 K12 (SEQ ID NO: 1) or hF8/N6K12RH (SEQ ID NO: 2).
12. The method of embodiment 1, wherein the lentiviral vector includes SEQ ID NO: 3 or SEQ ID NO: 4, or a functional variant thereof.
13. The method of embodiment any one of the previous embodiments, which is a method for treating a hemostasis related disorder in the subject, and the subject is in need of such treatment.
14. The method of any one of the previous embodiments, wherein the subject has hemophilia A (hemA), von Willebrand disease, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated vascular coagulation (DIC), or over-anticoagulation treatment disorder.
15. The method of any one of the previous embodiments, wherein the subject is a HemA subject with preexisting anti-FVIII inhibitory antibodies.
16. A method of treating a subject in need of plasma Factor VIII, essentially as described herein.
Previously described in vivo hematopoietic stem cell (HSC)-based, platelet-targeted gene therapy protocols partially corrected the bleeding phenotype in hemophilia A (HemA) mice for over five months. In that protocol, lentiviral vectors (LVs) carrying a factor III (FVIII) transgene (G-F8/N6-LV), driven by a megakaryocyte-specific promoter (Gp1bα), were delivered via intraosseous (IO) infusion to transduce HSCs. Maturation of transduced HSCs down the megakaryocyte-lineage led to ectopic FVIII production in platelets. However, an effective and clinically feasible protocol was still required to improve LV transduction efficiency and increase platelet-FVIII functionality.
To enhance LV transduction, this example describes a combined drug regimen of dexamethasone and anti-CD8α monoclonal antibody, followed by IO infusion of LVs in mice. In M-GFP-LV-treated (M=MND: ubiquitous promoter) C57BL/6 mice, a 2-fold increase in GFP+ bone marrow cell numbers was observed on day 7 and up to 14% of GFP+ HSCs on day 160. In G-F8/N6-LV treated HemA mice, significant improvement in phenotypic correction was seen on day 84.
In addition, to improve platelet-FVIII functionality, F8X10K12 (with increased expression and bioactivity compared to F8/N6) was incorporated into the LVs. Treatment with G-F8X10K12-LV in HemA mice produced a higher level of platelet-FVIII, however induced high-titer anti-FVIII inhibitors. This example also shows incorporation of F8/N6K12RH with enhanced bioactivity only to LVs. Following treatment with combined drugs and IO infusion of G-F8K12RH-LV, HemA mice showed significant phenotypic correction without anti-FVIII inhibitor formation. This protocol may provide a readily translatable treatment for hemophilia.
Thus, here, an effective and clinically feasible protocol was developed by improving LV transduction efficiency and increasing platelet-FVIII functionality. To enhance LV transduction, a combined drug regimen of dexamethasone and anti-CD8α monoclonal antibody was administered, followed by IO infusion of LVs. In M-GFP-LV-treated (M=MND: ubiquitous promoter) C57BL/6 mice. A 2-fold increase in GFP+ bone marrow cell numbers was observed on day 7 and up to 14% of GFP+ HSCs on day 160. In G-F8/N6-LV treated HemA mice, significant improvement in phenotypic correction was seen on day 84. To improve platelet-FVIII functionality, F8X10K12 with increased expression and bioactivity compared to F8/N6, was incorporated into the LVs. Treatment with G-F8X10K12-LV in HemA mice produced a higher level of platelet-FVIII, however induced high-titer anti-FVIII inhibitors. Further, following treatment with combined drugs and IO infusion of G-F8K12RH-LV (which has enhanced bioactivity), HemA mice showed significant phenotypic correction without anti-FVIII inhibitor formation. This protocol may provide a readily translatable treatment for hemophilia.
Introduction.
Inhibitory antibody formation against human factor VIII (FVIII) is a significant complication in treatment of hemophilia A (HemA) patients. Ectopic expression of FVIII in platelets could be an effective approach for treating HemA. Unlike circulating plasma FVIII, FVIII stored in α-granules of platelets is protected from being processed by antigen presenting cells, therefore significantly reducing the chance to induce anti-FVIII immune responses. Moreover, platelet FVIII is not neutralized by preexisting anti-FVIII inhibitory antibodies (inhibitors), which will benefit HemA patients who already developed inhibitors. During bleeding, the activated platelets locally excrete FVIII that can directly participate in the coagulation cascade and promote clot formation. It has recently been demonstrated that low levels of platelet FVIII can partially correct hemophilia phenotype in animal models.
One approach to direct FVIII long-term expression in platelets is intraosseous (IO) infusion of lentiviral vectors (LVs) carrying a FVIII transgene controlled by a megakaryocyte specific promoter Gp1bα (Gp1bα-F8-LVs) (Wang et al., Mol Therapy 23:617-626, 2015). In this in vivo gene therapy protocol of HemA, hematopoietic stem cells (HSCs) was efficiently transduced by LVs in situ, followed by FVIII transgene expression in the late stage of megakaryocyte differentiation, and finally storage of FVIII protein in α-granules of platelets. Previous work showed that a single IO infusion of Gp1bα-F8-LV resulted in over 3% platelets containing hFVIII, leading to partial phenotype correction in immune competent HemA mice with and without pre-existing inhibitors.
Furthermore, in vivo delivery of LVs can avoid many difficulties and potential toxicities encountered by ex vivo gene therapy including loss of stem cell properties and engraftment potential after cell transfer, negative effects of ex vivo cytokine stimulation and pre-conditioning of the subject. However, one potential limitation of in vivo gene therapy protocols is the induction of LV-specific immune responses, which may decrease LV transduction efficiency and eliminate LV transduced cells. It has been shown that dexamethasone (Dex) can suppress inflammatory responses after intravenous delivery of L Vs into the immune competent mice and increase LV transduction efficiency. It has also been shown that Rapamycin (Rapa) can enhance LV transduction efficiency for both in vivo and ex vivo gene therapy.
An optimized transgene for effective in vivo gene therapy of hemophilia would confer a higher gene expression level and a transgene product with enhanced biological activity. Since the size of a full-length human FVIII cDNA is quite large (>7 kbp), it cannot be easily packaged in viral vectors, often resulting in low titers of viruses. Therefore shorter cDNAs coding for FVIII variants were used in gene therapy preclinical research. Importantly, a B-domain deleted FVIII (BDD-FVIII) variant exhibits similar functional activity as the full-length FVIII (Pittman et al., Blood 81:2925-2935, 1993). Additional mutations were also incorporated into the gene coding for the BDD-FVIII to increase its secretion, such as, F8/N6 with a 226 aa B-domain variant sequence (Miao et al., Blood 103:3412-3419, 2004), codon optimized BDD-FVIII and F8/N6 (Ward et al., Blood 117:798-807, 2011), F8-V3 (McIntosh et al., Blood 121:3335-3344, 2013) with a 17-aa peptide coding sequence replacing the 226 aa/N6 in F8/N6, and F8-RH encoding a BDD-FVIII variant with R1645H (Siner et al., Blood 121:4396-4403, 2013). Some FVIII variants, like FVIII-RH and furin-cleavage site deleted BDD-FVIII variants (Nguyen et al., J Thromb Haemostas. 15:110-121, 2017), exhibit an increase in biological activity compared with BDD-FVIII, likely due to its slower dissociation of the A2-domain upon thrombin activation.
In this example, the therapeutic efficacy of HemA gene therapy was improve using IO infusion of LVs targeting FVIII expression in platelets. Immune competent mice were pretreated with Dex to suppress inflammatory responses and anti-CD8α monoclonal antibody (mAb) to inhibit cytotoxicity by transient depletion of CD8+ T cells. The pharmacologic intervention with combined Dex and anti-CD8 mAb treatment improved LV transduction efficiency and increased long-term transgene expression levels in mice. Furthermore, two new human FVIII (hFVIII) variants with higher expression and enhanced biological activity were also tested in immune competent HemA mice. The results demonstrated that combined drug treatment plus IO infusion of LVs containing the FVIII variant gene with enhanced biological activity significantly improved hemophilia phenotype correction.
Materials and MethodsAnimals: All mice were kept in a specific pathogen-free environment at Seattle Children's Research Institute (SCRI) according to National Institutes of Health guidelines for animal care and the guidelines of SCRI. The protocols were approved by the Institutional Animal Care and Use Committee at SCRI. HemA mice (factor VIII exon 16 knockout) with C57BL/6 (BL/6) genetic background were generated by crossing the mixed background HemA mice (SV129/BL6) with BL/6 mice for eight generations (Miao et al., Blood 114:4034-4044, 2009). BL/6 mice were purchased from the Jackson Laboratory. Only male HemA mice were used in this study.
Human factor VIII cDNA variants: Three human factor VIII cDNA variants were incorporated into the LVs, including B-domain variant of human factor VIII (hF8/N6) (Pipe, Haemophilia 15:1187-1195, 2009), hF8X10K12 (complete lentiviral construct: SEQ ID NO: 3), and hF8/N6K12RH (complete lentiviral construct: SEQ ID NO: 4). The sequences of the hF8X10K12 and hF8/N6K12RH inserts are provided in SEQ ID NOs: 1 and 2, respectively. Human F8 cDNA variants: Three human F8 cDNA variants were incorporated into the LVs, including B-domain variant of hFVIII (hF8/N6)22, hF8X10K1223,24 and hF8/N6K12RH19,24 (
Intraosseous infusion of lentiviral vectors: Lentiviral vectors were delivered into the mice by IO infusion as in previous studies (Wang et al., Mol Therapy 23:617-626, 2015; Wang et al., Blood 124:913-923, 2014; both incorporated herein by reference). Detailed information about the construction and production of lentiviral vectors and IO infusion of lentiviral vectors is provided below.
Immunosuppression treatment: Dex (MWIVet) and anti-CD8α mAb (BioXcell; clone YTS169.4) were used to transiently deplete innate immune responses and cytotoxicity, respectively. LVs were infused into the mice on day 0. Single drug pretreated mice were given Dex (100 mg/kg, −24 h, −4 h, 4 h and 24 h) or anti-CD8α mAb (4 mg/kg, day −1, 4 and 11) by intraperitoneal injection. Combined drugs pretreated mice were administrated with Dex (100 mg/kg, −24 h, −4 h, 4 h and 24 h) and anti-CD8α mAb (4 mg/kg, day −1, 4, 11, 16 and 21).
Characterization of transgene GFP/hFVIII expression: For M-GFP-LV treated mice, GFP expression in bone marrow cells of M-GFP-LV3 (LV carrying a GFP gene driven by the modified myeloid proliferative sarcoma virus (MND) promoter (Astrakhan et al., Blood 119:4395-4407, 2012; Challita et al., J. Virol. 69:748-755, 1995);
Assays for measuring hFVIII activities and anti-hFVIII antibodies: Plasma samples were isolated from the peripheral blood in experimental mice, which was obtained by retro-orbital bleeding. hFVIII activities were analyzed using a modified activated partial thromboplastin time assay (aPTT), and anti-hFVIII antibodies were measured by hFVIII Bethesda inhibitor assay as previously described (Miao et al., Blood 114:4034-4044, 2009; Kasper & Aronson, Thromb Diath Haemorrp 34:612, 1975).
Assays to characterize phenotypic correction in HemA mice: Correction of the bleeding phenotype of G-F8-LV treated HemA mice was evaluated using a modified tail clip assay, as previously described (Wang et al., Mol Therapy 23:617-626, 2015). The function of platelet-stored hFVIII in LV treated HemA mice was evaluated by Thrombin generation assay (TGA) using a TECHNOTHROMBIN® TGA Kit (Technoclone GmbH, Austria) and rotational thromboelastometry (ROTEM®) assay. The blood flow rate of the right carotid artery in G-F8-LV treated HemA mice after local damage was measured in a ferric chloride (FeCl3)-induced thrombosis model. The detailed procedures of TGA, ROTEM, the FeCl3 injury model are described below.
Statistical analysis: Data were expressed as mean ±the standard error of the mean (SEM). The statistical significance of the difference between means was determined using two-sample assuming unequal variances t test. Differences were considered significant at P less than 0.05.
Antibodies: Mouse hematopoietic lineage flow cocktail (Lineage) eFluor® 450, allophycocyanin (APC) anti-mouse CD117 (C-Kit) and Phycoerythrin-Cy7 (PE-Cy7) anti-mouse Ly-6A/E (Sca-1) were purchased from eBioscience. A polyclonal sheep anti-human factor VIII (SAF8C) was from Affinity Biologicals INC. A rabbit anti-sheep IgG antibody horseradish peroxidase (HRP) was from Bio-Rad Laboratories. Mouse anti-human factor VIII monoclonal antibody ESH-8 and GMA-012 were obtained from American Diagnostica Inc. and Green Mountain Antibodies, respectively. Goat anti-mouse Ig FITC was from BD Biosciences PharMingen.
Lentiviral vector construction, production and titration: Both pRRL⋅SIN⋅Gp1bα⋅hF8/N6⋅WPRE (pGp1bα-F8/N6) and pRRL⋅SIN·EF1α⋅hF8/N6·WPRE (pEF1α-F8/N6) were cloned in previous work (Wang et al., Mol Therapy 23:617-626, 2015). Then hF8/N6 in pGp1bα-F8/N6 and pEF1α-F8/N6 was replaced by hF8X10K12 and hF8/N6K12RH to create pRRL⋅SIN⋅Gp1bα⋅hF8X10K12⋅WPRE (pGp1bα-F8X10K12) and pRRL⋅SIN⋅Gp1bα⋅hF8X/N6K12RH⋅WPRE (pGp1bα-F8/N6K12RH), and pRRL⋅SIN⋅EF1α⋅hF8X10K12⋅WPRE (pEF1α-F8X10K12) and pRRL⋅SIN⋅EF1α⋅hF8X/N6K12RH⋅WPRE (pEF1α-F8/N6K12RH), respectively. pRRL⋅SIN⋅MND⋅eGFP⋅WPRE was obtained from the virus core at SCRI. All the reagents for cloning and isolating plasmids were purchased from Qiagen. The LVs (G-F8/N6-LV, G-F8X10K12-LV, G-F8/N6K12RH-LV, E-F8/N6-LV, E-F8X10K12-LV, and M-GFP-LV) were produced by the transient transfection of human embryonic kidney (HEK) 293T cells using polyethylenimine (PEI) and three plasmids including one transgene-LV construct, pMD2.G and pAX2G. Viral titers (ifu/ml) were determined by real-time quantitative PCR (qPCR) as described in previous studies (Wang et al., Mol Therapy 23:617-626, 2015; Auti et al., Embo Mol Med. 9:737-740, 2017).
HEK 293T cell transduction: HEK 293T cells were maintained in DMEM (Corning Cellgro) with 10% fetal bovine serum (FBS, Atlantic Biologicals), 2 mM L-glutamine (Corning Cellgro), 10 mM HEPES (Corning Cellgro) and 100 IU/ml Penicillin/100 μg/ml Streptomycin (Corning Cellgro). 5×104 cells were cultured in 200 μL medium and transduced with E-F8/N6-LV (MOI=100) and E-F8X10K12-LV (MOI=100) overnight with 4 μg/ml polybrene. After 24 h, 200 μL of culture medium was added. Cell were cultured for another 96 h, and collected to check FVIII expression via a flow cytometer (LSRII, BD Biosciences).
Intraosseous infusion of lentiviral vectors: The detailed intraosseous (IO) infusion procedure was described in the previous study (Wang et al., Mol Therapy 23:617-626, 2015). Briefly, the mouse was anesthetized with 2.5% isoflurane, and the hair around the right knee was shaved and the skin was sterilized and excised to expose the muscle layer. After twisting and carefully inserting a sterile 27-gauge needle fixed with an insulin syringe into the right tibia through the joint, 20 μL LVs was infused into the tibia at a speed of 10 μL/min via a new sterile 27-gauge needle connected with a microliter syringe (Hamilton). The infusion speed and volume were precisely controlled by a pump (NE-1010, New Era Pump Systems). The cut site was sutured after infusion.
Detailed methods of isolating bone marrow cells, white blood cells and platelets: Bone marrow cells were collected by flushing both femur and tibia of the mice with 5 ml of RPMI 1640. After lysing red blood cells with Ammonium-Chloride-Potassium (ACK) lysing buffer (0.15 M NH4Cl, 10 mM KHCO3, 1 mM EDTA, pH 8.1), other cells were obtained after filtering the medium through a filter with the pore size of 0.45 μm (Fisher Scientific). White blood cells and platelets were isolated from heparinized blood, which was collected by retro-orbital bleeding. White blood cells were collected after lysing red blood cells with ACK buffer. For platelets, 140 μL of heparinized blood was added with 200 μL of RPMI 1640, and the mixture was carefully transferred to the top of 300 μL of Fico/Lite™-Platelets (Atlanta Biologicals) and spin at 100 g for 10 min. Then carefully removed the clear upper solution and collected the cloudy middle layer to spin at 1,000 rpm for 5 min. Small white platelet pellets were washed by FACS buffer (PBS, 5% bovine serum albumin) twice.
Real-time qPCR: Real-time qPCR was performed on genomic DNA (gDNA), which was extracted from the isolated white blood cells and bone marrow cells in experimental mice using DNeasy Blood & Tissue Kit from Qiagen. gDNA was subject to real-time qPCR using two primer/probe sets (one for integrated lentiviral vector sequence and one for murine housekeeping β-actin sequence) and TaqMan Universal PCR Master Kit (Applied Biosystems) and run on Step One Plus real-time PCR System (Applied Biosystems). Murine copy number was determined by threshold cycle, which was compared to standard curves generated using gDNA isolated from single lentiviral copy A20 cell clones.
Detailed methods of thrombin generation assay: Firstly, both the protocol for Calibration Curve measurement and the protocol for sample measurement were created in PerkinElmer® Wallac Manger. Then the whole blood was drawn from the inferior vena cava (IVC) of the anesthetized mice into 3.8% sodium-citrate solution. Platelet-rich plasma (PRP) was obtained by centrifuging for 5 minutes at 100×g and carefully pipetting the top layer. Platelet-poor plasma (PPP) from untreated HemA mice was collected by centrifuging PRP for 10 minutes at 1,500 g. Platelet concentration in PRP was measured by a qNaono (iZON Science). 30 μL of PRP samples mixed with 10 μL of HemA PPP was added into black NUNC Maxisorp (Fisher). The plate was read immediately in a Victor 3 instrument (Perkin-Elmer) after adding 50 μL of TGA substrate (SUB). In a separate calibration measurement, 40 μL of thrombin calibrator at four different dilutions (1:2, 1:4, 1:20, 1:200) were combined with 50 μL TGA SUB and thrombin generation was detected for 10 min in 30 sec intervals at 37° C. Lag phase time, peak thrombin and the area under the curve (AUC) were obtained using the evaluation software provided by Technoclone.
Blood flow rate measurement: The experimental mice were anesthetized by 2.5% isoflurane. An incision was made with a scalpel directly over the right common carotid artery, and a segment of the artery was exposed using blunt dissection. A miniature Doppler flow probe (Model 0.5 VB, Transonic System Inc., Ithaca, N.Y., USA) was attached to the carotid artery to monitor blood flow. Blood vessel damage was induced by applying two pieces of filter paper (1×2 mm) saturated with 20% Ferric chloride (FeCl3) solutions. The pieces of filter paper were placed on the top of carotid artery in contact with the adventitial surface of the vessel. After 2-min exposure, the filter paper was removed and the vessel was washed with sterile normal saline. Carotid blood flow was continuously monitored for 30 min after FeCl3 application. Wild-type C57BL/6J mice and HemA mice were used as positive and negative controls, respectively. Average blood flow rate over 30 minutes after FeCl3 treatment was used to evaluate the extent of protection from occlusion. The mice were sacrificed after measurement.
ResultsComparison of GFP expression in immunocompetent and deficient mice following IO delivery of M-GFP-LV To evaluate if immune responses impact transgene expression, M-GFP-LV (1.1×108 infectious units (ifu)/animal) was intraosseously delivered into immune deficient Rag2−/− mice and immune competent BL6 mice. 7 days after infusion, GFP expression levels in total bone marrow cells and HSCs in Rag2−/− mice were similar to that in BL6 mice (
Pharmacological approach to enhance GFP expression in immune competent mice following with IO delivery of M-GFP-LV Based on the results obtained from LV-treated immune-competent and deficient mice, whether pharmacological agents designed to non-specifically or specifically block these immune responses will limit clearance of transduced cells, leading to more persistent and higher levels of sustained transgene expression, was explored. Firstly, BL6 mice were treated Dex with (100 mg/kg, −24 h, −4 h, 4 h and 24 h) before and after IO infusion of M-GFP-LV (8.8×108 ifu/animal, day 0). 7 days after LV infusion, the Dex+LVs treated mice produced significantly higher GFP expression levels in both total bone marrow cells and HSCs than those in LVs only treated mice (
Secondly, CD8+CD3+ T cells were transiently depleted by IP injection of anti-CD8α mAb to suppress cytotoxic function (
In order to increase LV transduction and preserve survival of transduced cells, a short course of combination drug treatment with Dex (100 mg/kg, −24 h, −4 h, 4 h and 24 h), and anti-CD8α mAb (4 mg/kg, day −1, 4, 11, 16 and 21) before and after IO infusion of M-GFP-LV (3.6×108 ifu/animal) was administered. 7 days after IO infusion, GFP expression levels in total bone marrow cells in drugs+LVs treated mice was significantly higher than that in LVs only mice. Whereas, GFP expression level in HSCs was similar in these two groups of mice probably because HSC transduction reached saturation with relatively high dose of LVs. In the long term, GFP expression levels in both total bone marrow cells and HSCs in Drugs+LVs treated mice were significantly higher than that in LVs only treated mice (
Pharmacological approach to improve phenotype correction in immune competent HemA mice with IO delivery of G-F8/N6-LV. Next, the same pharmacological approach was applied to improve the therapeutic effects of IO infusion of G-F8/N6-LV in HemA mice. HemA mice were given with the combined drug treatment (Dex+anti-CD8α mAb) plus IO infusion of G-F8/N6-LV (2.2×106 ifu/animal). 10 weeks after IO infusion, hemophilia phenotype correction was evaluated with two injury mouse models: tail clipping and FeCl3-induced thrombosis. In the tail clipping assay, the LVs only or drugs+LVs treated mice had significantly reduced blood loss than HemA control mice (
F8X10K12 with higher expression and higher functional activity combined with drug treatment improved hemophilia A phenotypic correction. To improve therapeutic effects of in vivo gene therapy of HemA mice, cDNAs encoding FVIII variants with high expression and functional activity were next incorporated. F8X10K12 was a novel human FVIII cDNA variant with a deleted B-domain, mutations in A1 domain to enhance secretion (X10), and mutations in Cl and C2 domains to increase functional activity (K12) (
Next, G-F8X10K12-LVs (
The long-term therapeutic efficiency of combined drugs+LVs in HemA mice was evaluated by a tail clipping assay. On day 120 after IO infusion of LVs, all three groups of mice treated with F8X10K12-LV+drugs, or G-F8X10K12-LV only, or G-F8/N6-LV only, showed significantly reduced blood loss compared with HemA control mice (
The functional activity of platelet-FVIII was also evaluated in vitro by thrombin generation assay using PRP isolated from HemA mice. Although platelets play a major role in the burst of thrombin generation in clotting cascade, FVIII stored in platelets enhances this process to promote fibrin clot formation. Compared to HemA controls, wild-type controls had shorter lag phase time, higher peak height thrombin and higher total generated thrombin (area under the curve (AUC) (
F8/N6K12RH with higher functional activity combined with drug treatment significantly enhanced hemophilia A phenotyping correction Even though relatively higher platelet-FVIII level in G-F8X10K12-LV treated HemA mice enhanced blood clotting, high-titer inhibitory antibodies were induced due to secretion of FVIII into the circulation. Thus F8/N6RHK12 (
G-F8/N6K12RH-LV was generated and delivered into HemA mice with combined drug treatment. As expected, the average platelet-FVIII antigen levels in G-F8/N6K12RH-LV-only and G-F8/N6-LV-only treated mice were similar. However, the platelet-FVIII levels in G-F8/N6K12RH-LV+drugs-treated mice were significantly higher than that in G-F8/N6K12RH-LV-only and G-F8/N6-LV-only treated mice on day 84 (
HemA phenotype correction of G-F8/N6-LV only or G-F8/N6K12RH-LV+drugs treated mice was also evaluated by measuring carotid artery blood flow rate on day 84 (
The whole blood samples were collected on day 140 to evaluate the platelet FVIII function using a ROTEM assay. Each assay was carried out for 1 hr to obtain values of clotting time (CT), clot formation time (CFT), α-angle, and max clot firmness (MCF). Statistical significance was analyzed based on the relevance to the G-F8/N6-LV treated mouse group. *<0.05, **<0.01, ***<0.001.
Discussion. Current clinical trials for hemophilia A or B gene therapy using recombinant adeno-associated viral (AAV) vectors (rAAV) have shown very promising results. However, lentiviral gene therapy is superior in several aspects. First, AAV persists as episomal, concatemerized vector following in vivo gene transfer. Overtime, transgene expression may decrease, and repeated dosing will be required. It remains to be shown whether repeated dosing is feasible, or effective. In contrast, lentiviral vectors are an integrating vector that is incorporated into the genome of transduced cells. If successfully integrated into long-lived bone marrow HSCs, a single treatment of LV-mediated gene therapy may be sufficient for life-long therapeutic benefit. In addition, In contrast to AAV which has a transgene capacity of 4.5 kb, LV can easily accommodate much larger genes such as FVIII cDNA. LV can efficiently transduce both dividing and non-diving cells, leading to efficient transduction of primitive HSCs. Furthermore, recent clinical trials of ex vivo HSC gene therapy have shown remarkable therapeutic benefits and safety records for patients with severe genetic diseases including X-linked severe combined immunodeficiency, Wiskott-Aldrich syndrome, spinal muscular atrophy, and sickle cell disease Most significantly, genotoxicity by clonal dominance or significant clonal expansions from vector integration were not observed in these patients.
In the current study, the developed gene therapy protocol for HemA aims to treat HemA patients by IO infusion of LVs containing FVIII cDNA under expression control by a megakaryocyte-specific promoter, which leads to expression of the gene in the platelets. This gene therapy protocol has several advantages over existing protocols that include the ex vivo gene therapy protocol targeting HSC transduction or the in vivo gene therapy protocol targeting FVIII expression in hepatocytes. Compared with ex vivo gene therapy, IO infusion could efficiently in situ transduce HSCs to avoid the lengthy and complicated procedures of cell manipulation and transplantation. Significantly, the herein described approach avoids the requirement for use of fully or partially myeloablative conditioning regimens required for successful ex vivo gene therapy.
Compared to other in vivo LV-mediated gene therapy approaches, IO delivery is unique. Slow injection (e.g., at a rate of 2 μL/min to 13 μL/, or at 10 μL/) of LVs into bone marrow cavity can minimize or even avoid the systemic toxicity caused by intravenous delivery of LV, AAV and other candidate gene therapy vectors. In addition, compared with hepatocyte-targeted LV gene transfer which induced high risk of antibody formation after FVIII is secreted from hepatocytes into circulation, the platelet-targeted FVIII gene therapy generated FVIII stored in platelets and protected from processing by antigen presenting cells and neutralization by anti-FVIII inhibitors. During bleeding, FVIII was released by activated platelets on-site to efficiently enhance clot formation. Bleeding phenotype is partially corrected in HemA mice with or without pre-existing anti-FVIII inhibitors. Additionally, targeting factor VIII expression to platelets for HemA gene therapy did not increase thrombotic risks but promoted immune tolerance in HemA mice. Therefore, the described strategy of IO LV delivery targeting FVIII expression in platelets is highly promising to treat HemA patients with or without inhibitors.
However, in vivo LV gene therapy brought associated immune responses that may decrease LV transduction efficiency and eliminate LV transduced cells. In immune competent mice given IO infusion of MND-GFP-LV, the percentage of GFP+HSCs decreased over time (
A modified FVIII cDNA producing FVIII protein with higher expression level, enhanced bioactivity and longer half-life would be desirable for successful gene therapy of hemophilia A. Compared with human FVIII, canine FVIII is functionally more active and porcine FVIII expresses at higher levels. Novel recombinant FVIII cDNAs are thus designed with mutations at specific sits replacing with porcine- or canine-corresponding amino acids to increase secretion and functionality on the basic frame of human VIII sequence. Compared with F8/N6, F8X10K12 directed by a ubiquitous promoter produced a 25-fold increase of FVIII activity in HemA mice following hydrodynamic injection (
It has been reported by that high levels of FVIII expression in platelets could induce platelet apoptosis. Thus, the described platelet-targeted gene therapy of hemophilia A focused further on enhancing bioactivity rather than expression levels. The cDNA of a FVIII variant, F8/N6K12RH with higher activity contributed by a R1645H mutation (Siner et al., Blood 121:4396-4403, 2013) and K12 mutations in light chain, was incorporated into the LV construct. Compared with F8/N6, F8/N6K12RH directed by a ubiquitous promoter produced a 2.7-fold increase of FVIII activity in HemA mice (
FVIII ectopic expression in platelets provides an effective pathway for gene therapy of HemA patients, especially for patients with pre-existing anti-FVIII inhibitors as a result of frequent intravenous infusion of FVIII. FVIII stored in platelets is not only protected from being neutralized by inhibitors in peripheral blood, but also significantly improves the bleeding phenotype even with as low as 1%-5% of peripheral blood platelets containing FVIII. The therapeutic effects of platelet-FVIII was characterized by several FVIII function assays including in vitro assays such as chromogenic assay (Shi et al., Mol Gen Metabol. 79:25-33, 2003; Baumgartner et al., J Thromb & Haemostas. 13:2210-2219, 2015) and thrombin generation assay (Monroe et al., Arterios Thromb Vas Biol. 22:1381-1389, 2002), and in vivo assays such as tail clipping assay (Wang et al., Mol Therapy 23:617-626, 2015; Shi et al., J Clin Invest 116:1974-1982, 2006) and FeCl3 carotid artery thrombosis assay (Yarovoi et al., Blood 105:4674-4676, 2005; Yarovoi et al., Blood 102:4006-4013, 2003). FVIII-containing platelets were isolated for use in in vitro assays. Isolated FVIII-containing platelets were lysed, and released FVIII functional activity was detected by a chromogenic assay using a Coatest FVIII:C/4 kit. However, results from the chromogenic assay could not accurately predict therapeutic efficacy because of the very low level of platelet-FVIII even in transgenic mice (the highest platelet-FVIII level of 11 mU/108 platelets in LV18tg+/+ mice, described in Baumgartner et al., J Thromb & Haemostas. 13:2210-2219, 2015). In place of the chromogenic assay, the thrombin generation assay was used where FVIII-containing platelets were directly added into HemA PRP and function was expressed as three parameters (lag phase time, peak height thrombin and total generated thrombin). LV treated mice had shorter lag phase time compared to HemA controls, suggesting that FVIII-containing platelets played an important role in the early stage of clot formation by accelerating thrombin generation. The tail clip assay of treated mice revealed IO infusion of LVs significantly corrected bleeding phenotype compared to HemA control mice. However, therapeutic effects between a combined drug regimen+LVs treatment and LVs only treatment could not be distinguished with this assay. Therefore, the FeCl3 carotid artery thrombosis assay, where blood flow rate was monitored after local damage to the carotid artery, was used. Compared with the modified tail clipping assay, FeCl3 induced carotid thrombosis appears to be more sensitive to monitor the role of platelet-FVIII in enhancing local blood clot formation in arteries, and capable of distinguishing therapeutic differences in the two treatment groups. Results from this assay showed that platelet-FVIII was effective in arterial clotting and that a combination drug regimen with new FVIII variant, F8/N6RHK12, achieved phenotypic correction of HemA in mice.
In conclusion, persistent FVIII gene expression in platelets was achieved by IO infusion of G-F8/N6K12RH-LV into HemA mice pretreated with a combination of Dexamethasone and anti-CD8α monoclonal antibody. Transient suppression of immune responses to LVs and LV-transduced cells increased LV transduction efficiency and produced long-term stable transgene expression. F8/N6K12RH with an enhanced bioactivity effectively improved the therapeutic effect of FVIII containing platelets in phenotype correction assay. The function of platelet-FVIII isolated from LV-treated mice was confirmed using a thrombin generation assay. Phenotypic correction of hemophilia was demonstrated using blood clotting assays with tail clip and carotid artery injury models. This approach may provide a readily translatable treatment for hemophilia in large animals and clinical trials.
Example 2. Intraosseous Delivery of Lentiviral Virus Acts as a Therapeutic Strategy for In Vivo Gene Therapy of HemophiliaAs discussed above, lentiviral vectors (LVs) delivered by intraosseous (IO) infusion at a precisely controlled speed can efficiently transduce bone marrow hematopoietic stem cells (HSCs) in mice. IO infusion of LV carrying a human FVIII/N6 transgene driven by a platelet-specific Gp1bα promoter into hemophilia A (HemA) mice produced FVIII stored in platelet α-granules. These platelet FVIII partially corrected the bleeding phenotype over five months in HemA mice with or without pre-existing anti-FVIII inhibitors.
In the current example, IO delivery of LVs was applied in humanized NSG mice to establish a translational research model for in vivo gene therapy of hemophilia. Firstly, whether high level transgene expression could be achieved in human megakaryocytes (Megs) was examined. Human CD34+ cells were transduced with Cocal-MND-GFP-LV (M-GFP-LV, MOI=25) or Cocal-Gp1bα-GFP-LV (G-GFP-LV, MOI=25) and then cultured with Megakaryocyte Expansion Supplement (MES). 14 days after LV transduction, high level of GFP expression (36%) was detected in both M-GFP-LV transduced Megs (CD41+ cells) and non-Megs (CD41− cells). Furthermore, in G-GFP-LV-transduced human cells, GFP specifically expressed in Megs, but not in other types of cells derived from HSCs.
Next, FVIII specific expression in human Megs was evaluated in Cocal-GP1bα-F8/N6-LV (G-F8/N6-LV) transduced human CD34+ cells. FVIII expression level in G-F8/N6-LV transduced human cells increased from 7% to 28% when the LV doses increased from MOI=10 to 50 on day 21 after LV transduction. These results indicated that platelet-specific promoter, Gp1bα, could specifically guide high level transgene expression in human Megs.
Transgene expression in humanized NSG mice following IO delivery of LVs was also investigated. Humanized NSG mice were created by I.V. infusion of 1×106 human CD34+ cells into 6-week female NSG mice. After 8 weeks, G-GFP-LV, G-F8/N6-LV and Cocal-GP1bα-F8X10-LV (G-F8X10-LV) were delivered into the humanized mice by IO infusion and transgene (GFP or human FVIII) expression was evaluated in human platelets. However, human platelets in blood of humanized mice rapidly decreased to undetectable levels following human cells engraftment, possibly due to recognition and destruction by murine macrophage cells.
In order to examine transgene expression in human platelets, the mice were given a series of clodronate liposome treatments (CLT) (0.7 mg/animal, Day 0, 2, 7, 13), which was reported to transiently deplete murine macrophage cells. CLT treatment induced recovery of human platelets from an undetectable level to the level comparable to the percentage of engrafted human cells in total PBMCs. In addition, similar level of GFP expression was detected in both human and murine platelets of CLT+G-GFP-LV treated mice. Most importantly, higher platelet FVIII level was found in CLT+G-F8/N6-LV or G-F8X10-LV treated mice than G-F8/N6-LV or G-F8X10-LV only treated mice. Over 10% of the human cells expressed FVIII when human CD34+ cells were isolated from the G-F8/N6-LV-treated humanized mice and cultured with MES for 6 days. Next, LV integration sites in isolated human cells were examined by LAM-PCR.
Based on the described studies, IO infusion of LVs can be a valuable strategy for in vivo gene therapy of hemophilia A and other blood clotting disorders that would benefit from the expression of FVIII.
Example 3. Intraosseous Delivery of Lentiviral Virus Produces Human Platelet-Specific Factor VIII in Humanized NSG MiceThis example establishes proof-of-principle research models in humanized NSG mice for translational applications of this novel strategy in human clinics.
Hemophilia A (HemA) with a deficiency of functional plasma factor VIII (FVIII) is an ideal disease candidate for gene therapy to attain long-term therapeutic FVIII levels.
Described herein is a new clinically translatable strategy for treating HemA. In this strategy, self-inactivating lentiviral vector (LV) carrying FVIII transgene driven by a platelet-specific promoter Gp1bα (G) (G-FVIII-LV) was delivered into HemA mice without preconditioning via intraosseous (IO) administration. G-FVIII-LV can effectively transduce hematopoietic stem cells (HSCs). Then, FVIII specially expressed and stored in platelet α-granules can partially correct bleeding phenotype over five months in immune-competent HemA mice with or without pre-existing anti-FVIII inhibitors (Wang et al., Mol. Ther., 23(4):617-626, 2015).
Human CD34+ cells were cultured in SFEM II supplemented with cytokine cocktail for expansion of human hematopoietic cells (CC110), or serum-free culture supplement for expansion of human megakaryocytes (Meg) for 7 and 13 days (
6-week NSG mice were retro-orbitally injected with 1×106 human CD34+ cells one day after preconditioning with busulphan (25 mg/kg). After 13 weeks, bone marrow (
M-GFP-LV was intraosseously infused into humanized NSG mice, which were generated by retro-orbital injection of human CD34+ cells 9 weeks ago. Four weeks later, the mice were sacrificed and bone marrow and spleen were isolated. GFP expression in total cells, human CD45+ and murine CD45+ cells in bone marrow (
G-CSF mobilized human CD34+ PBSCs cultured with SFEM II+Meg successfully differentiated into megakaryocytes in vitro, and transgene expressed in human cells after LV transduction.
Humanized mice were generated by retro-orbital injection of G-CSF mobilized human CD34+ PBSCs into preconditioned NSG mice. In the long term, human CD34+ cells successfully renewed and differentiated into hematopoietic lineage cells in vivo.
GFP expressed in human cells of humanized NSG mice after intraosseous infusion of LVs. Next, humanized NSG mice will be treated with intraosseous delivery of FVIII-LVs.
The IO delivery of LVs technology was applied in humanized NSG mice to establish a translational research model for in vivo gene therapy of hemophilia. Efficient transduction of human CD34+ cells to produce megakaryocyte-specific FVIII expression by G-F8-LV was demonstrated. In addition, it was demonstrated that following IO delivery of G-F8/N6-LV into humanized NSG mice, human CD34+ cells isolated from the treated mice showed significant FVIII expression. LV integration sites in isolated human cells can be examined by LAM-PCR, confirming the safety of this strategy. These results further confirm that IO infusion of LVs could be a valuable strategy for in vivo gene therapy of hemophilia A.
Example 4: Evaluation of the Efficacy and Safety of Intraosseous Delivery of Platelet-Specific Factor VIII-Lentiviral Vectors as an In Vivo Gene Therapy for Hemophilia in Humanized NSG MiceHemophilia A is a genetic disorder resulting in a deficiency in functional factor VIII (FVIII) secreted in plasma and presents as an ideal disease model for gene therapy. As described above, an in vivo intraosseous injection (IO) therapy has successfully developed to deliver FVIII/N6 lentivirus (G-F8VIII-LV) driven by the platelet-specific Gp1bα promoter. Viral transduction of hematopoietic stem cells (HSCs) ensures long-term continued maintenance of the gene therapy. FVIII is then produced and stored in the α-granules of platelets, avoiding detection by the immune system, making this therapy accessible to patients who have already developed inhibitory antibodies to FVIII. IO delivery of G-F8-LV partially corrected the hemophilia phenotype in mice for at least 5 months. The aim of the current study was to assess the safety and efficacy of IO delivery of G-FVIII-LV in humanized mice to assure the feasibility of translating this therapy to clinical models.
In vitro studies were first conducted to confirm successful transduction and appropriate expression of G-F8-LV and G-GFP-LV in human CD34+ cells isolated from G-CSF mobilized donors. Cells were cultured in StemSpan™ Serum-Free Expansion Medium (SFEM) supplemented either with cytokine cocktail (CC110) to expand hematopoietic stem cells or with Megakaryocyte expansion supplement (MEG) following transduction to promote differentiation into megakaryocytes. After 7 days of growth, cells cultured in MEG had a higher percentage of both CD41a+ and late-stage differentiated CD41a+CD42b+ megakaryocytes than those cultured in CC110, indicating preferential differentiation into megakaryocytes in this media. Higher FVIII and GFP expression was also observed in MEG cultured cells, suggesting successful megakaryocyte-specific expression, as directed by the Gp1bα promoter.
These LVs were then tested in vivo in humanized NSG mice. NSG mice with and without the W-41 mutation in the c-Kit gene were preconditioned with busulfan (25 mg/kg) 24 hours before the transplantation of hCD34+ cells via retro-orbital (RO) injection. Eight weeks later, higher engraftment of hCD34+cells was observed in the W-41 NSG cohort compared with NSG mice, averaging greater than 80% engraftment in bone marrow. Human platelet count was also higher in the W-41 NSG mice.
IO delivery of LVs was next performed in cohorts of transplanted mice. Clodronate liposome treatments increased human platelet counts, allowing for detection of notable FVIII expression in platelets isolated from PBMCs of mice treated with G-FVIII-LV. Bone marrow was isolated several weeks after the IO injection, and engrafted human HSCs were isolated using the AutoMACS CD34 MicroBead Kit. The isolated cells were subsequently cultured in MEG and expressed significant levels of FVIII and GFP after 14 days.
The genomic DNA (gDNA) of hCD34+ cells transduced by G-GFP-LV and G-F8-LV respectively was isolated at various MOls and the bone marrow of two groups of mice that had undergone IO injections with these two viral vectors respectively, and marking number was assessed by qPCR.
Using LAM-PCR, integration sites of the lentivirus are being analyzed to assess safety of this therapy. It is expected that there will be no clonal expansion. The integration sites are predicted to be random and polyclonal.
Given its efficacy in humanized mouse models, IO delivery of platelet-specific FVIII presents as a promising in vivo gene therapy for correcting the hemophilia phenotype.
Example 5. Correction of Canine Hemophilia A Via Intraosseous Delivery of a Platelet-Specific Factor VIII-Lentiviral VectorThis example describes development of a preclinical protocol of treatment of HemA dogs using interosseous (IO) delivery of lentiviral vectors (LVs).
Background: Hemophilia A (HemA) results from a factor VIII (FVIII) deficiency. Traditional protein infusion to treat HemA is costly, inconvenient, and short-term. Intraosseous (IO) gene therapy via delivery of lentiviral vectors (LVs) into bone marrow targeting FVIII expression in platelets successfully treated HemA mice with or without inhibitors. To facilitate the translation of this novel strategy to clinical application, treatment of HemA dogs via IOgene therapy was investigated.
Methods: The VSVG-pseudotyped G-cFVIII-LV incorporating a platelet-specific promoter Gp1bα and canine FVIII gene at either 2.51*108 ifu/kg or 1.34*109 ifu/kg was injected into tibia bones of HemA dogs M80 and R04 separately. The injection volume of LV was 4.5 ml for each dog. Gene expression of cFVIII was examined in platelets and plasma isolated from LV-treated dogs by ELISA and aPTT assays. The correction of HemA phenotype was evaluated by whole blood clotting time (WBCT) and thromboelastography (TEG) testing.
Results: Canine FVIII can be detected in platelets with the highest expression at 30 mU/108 platelets (
Conclusion: This example establishes a representative IO gene therapy protocol to treat HemA mammals (exemplified by dogs) successfully. This study provides a strategy for safe and effective application of gene therapy in vivo for treating HemA patients.
Example 6: Intravenous Delivery of Lentiviral Vectors to Treat Hemophilia A in a HSCs Mobilization Mouse ModelThis Example describes development of a novel in vivo approach to genetically modify hematopoietic stem cells (HSCs) in peripheral blood by lentiviral vectors (LVs) for the treatment of Hemophilia A.
Primitive HSCs entering peripheral blood from bone marrow (BM) were first mobilized by administering a combination of G-CSF and AMD3100 in mice, which was followed by intravenous injection (IV) of lentiviral vectors carrying FVIII gene (LVs). Gene delivery efficiency was then investigated by using four types of Lentivirus Vehicles (LVs) encoding the GFP or FVIII gene, either driven by ubiquitous promoters or by a megakaryocyte specific glycoprotein-1 bα (Gp1bα) promoter (MND-GFP-LV, G-GFP-LV, EF-FVIII-LV, G-FVIII-LV). The results show that a single IV injection of LVs could significantly increase transduced HSCs in bone marrow of HSCs mobilized mice. Meaningfully, IV delivery of G-FVIII-LVs achieved long-term FVIII expression in platelets and corrected the hemophilia A phenotype partially without signs of inhibition of FVIII expression. Therefore, the in vivo lentivirus delivery strategy has high potentials for an effective treatment of FVIII deficiency in patients.
Introduction: Hemophilia A with deficiency of FVIII is a rare disease as defined by World Health Organization. Hemophilia A (HemA) is a serious bleeding disorder caused by deficiency in Factor VIII gene. Individuals with hemophilia A suffer from repeated bleeding episodes, which can cause chronic joint disease. After expensive treatment with the recombinant active FVIII, 30% of individuals develop an immunogenic response in the form of neutralizing antibodies against FVIII (Miao, Thrombosis Journal 14:41, 2016).
Gene therapy is a good strategy to treat HemA (Roth et al., New England Journal Med 344:1735-1742, 2001; White, Thrombosis and Haemostasis 86:172-177, 2001; Naldini, Nature 526:351-360, 2015; Yarovoi et al., Blood 102:4006-4013, 2003). Recently, clinical trials by biotechnology companies show very promising results. AAV vectors are used to delivery FVIII, hybrid liver-specific promoter (HLP) specifically driving its expression in liver for restoring FVIII in plasma (Pasi et al., Haemophilia 22:151-152, 2016). However, there is a risk of triggering immune responses to generate anti-FVIII inhibitory antibody, which eventually results in reducing/eliminating of the FVIII in plasma (Ertl et al., Human Gene Therapy 28:328-337, 2017; Doering et al., Molecular Therapy: J Amer Soc Gene Therapy 15:1093-1099, 2007). In addition, quite many patients have developed inhibitors against AAV vectors, thus that such gene therapy method will not work.
Specific expression of FVIII in platelets is a decent gene therapy method for the treatment of HemA. Wang et al delivered lentivirus construct containing the Gp1bα-FVIII via intraosseous infusion (Molecular Therapy: J Amer Soc Gene Therapy 23:617-626, 2015). Blood clotting can be partially corrected by the treatment with platelets specific expression of the FVIII. There are also other investigators who employ Gp2bα promoter to drive FVIII expressions. Both of the methods resulted in FVIII storing in platelets. However, the mice with LVs intraosseous delivery or transplantation of transuded HSCs didn't show FVIII inhibitors.
Using G-CSF and AMD3100 to mobilize primitive progenitor cells is an efficiency method of enforcing the HSCs in bone marrow into peripheral blood. Many studies confirmed that mobilized HSCs can be transduced by an integrating adenovirus vectors in vivo. Additionally, some preclinical studies use the mobilization to enhance gene therapy efficacy. For example, others employed immune reconstitution in mobilized dogs with X-linked severe combined immunodeficiency disease in mobilization dogs. Combination mobilization and IV delivery lentivirus vectors is a good strategy. Therefore, the hypothesis of this study is that mobilized HSCs from bone marrow to peripheral blood could be transduced by lentivirus carrying GFP or FVIII gene. Furthermore, platelets-specific promotor driving FVIII expression in platelets could avoid the development of FVIII inhibitors.
Materials and MethodsAnimals and Cells: All in vivo studies and HemA breeding have been approved by the Institutional Animal Care and Use Committee (IACUC) of Seattle Children's Hospital, under IACUC Protocol No. 130001330. All animal procedures and animal care were performed according to institutional animal research guidelines and were also in compliance with Seattle Children's Hospital Animal Management Regulations. HEK293T cells were obtained from the ATCC. Cells were cultured in relevant cell culture medium supplemented with 10% (vol/vol) FBS, 1% (vol/vol) P/S, 1% (vol/vol) Glutamine and 1% (vol/vol) HEPES. Testing for potential mycoplasma contamination was done by ELISA.
Mice mobilization and in vivo transduction: Male black/6 mice or male HemA mice (6-8 weeks) are subjected to mobilization procedure. C-GSF was subcutaneous injected 50 ng/mouse for 4 days. At the fifth day, AMD3100 was injected in mice (5 mg/kg) via SC. Lentivirus infused at the fifth day after AMD3100 injection 30 minutes. Dexamethasone was dosed to mice intraperitoneally 16 hours and 2 hours before lentivirus injection. See
Lentivirus production: Lentiviral vectors (LVs) were produced as described previously (Wang et al., Mol Ther 23(4)617-626, 2015); briefly 12 μg Transgenes (pRRL-SIN-MND-eGFP-WPRE, pRRL-SIN-Gp1bα-eGFP-WPRE, pRRL-SIN-EF-1α-hFVIII/N6-WPRE, and pRRL-SIN-Gp1bα-hFVIII/N6-WPRE), 6 μg viral package plasmid (psPAX2), 3 μg of viral envelop plasmid (pMD2·G) were transfected HEK293T cells with 33 μg of polyethyleneimine (linear, MW ˜40,000, Polysciences, Inc, Warrington, Pa.). LV-containing supernatants were concentrated ˜2000-fold by low-speed (8000 g) centrifugation for 16-18 hr at 4° C. Infectious vector stocks were stored at −80° C. Viral titers were determined by real-time quantitative PCR (qPCR) as described previously (Kerns et al., Blood 115:2146-2155, 2010). Briefly, 2×105 Nalm-6 pre-B cells were transduced with serial dilutions of concentrated virus. Cells were harvested 7 days later, washed twice with PBS and used for genomic DNA (gDNA) extraction. Viral titers (ifu/ml) were calculated from the transduction rate determined by gDNA qPCR. Average titers were 2.2×109 ifu/ml, 2.5×109 ifu/ml, 2.5×109 ifu/ml, and 1.1×109 ifu/ml for M-GFP-LV, E-F8-LV, G-GFP-LV, and G-F8-LV, respectively.
Flow cytometers: HSCs or LSKs were stained and gated by lineage—ckit+scal+ in BM cells or PBMCs. Platelets were isolated from PBMCs by centrifuging method. The platelets were separated by isolation buffer Fico/Lite™-Platelets (Atlanta Biologicals) after 90 g/min, 8 min spinning. The isolated platelets were stained by CD42d. B, T, and myeloid cells were stained by surface maker B220, CD3, and CD11b (eBioscience San Diego, Calif.). For FVIII expression in platelets, after surface staining, platelets were fixed by fix and perm buffer (BD). Mouse anti-hFVIII monoclonal antibody (ESH8, American Diagnostica, Seabrook, Tex.) was used for primary antibody, followed by second antibody secondary goat anti-mouse Ig FITC (BDBiosciences). Flow cytometric analysis was conducted using LSRII (BD Bioscience) and the data were analyzed using FlowJo (Ashland, Oreg.).
FVIII activities and FVIII antibody assays in Plasma: hFVIII activities in mouse plasma were analyzed using aPTT, and anti-hFVIII antibodies were measured by hFVIII Bethesda inhibitor assay as previously described (Miao et al., Blood 114:4034-4044, 2009; Kasper et al., Thromb Diath Haemorrh 34:612, 1975).
Clotting functional assay: Rotation thromboelastometry (ROTEM®) functional clotting assay was employed to characterize the blood clotting function, using a computerized ROTEM® four-channel device (Instrumentation Laboratory, USA). Mice blood was collected via chin bleeding and thoroughly mixed with 10% sodium citrate. ROTEM® assays were performed under Intern program by the mini cup protocol. The blood collected from control and gene therapy groups were subjected to run for one hour to investigate coagulation time (CT), clot formation time (CFT), α-angle, and max clot firmness (MCF) on ROTEM®.
Copy number detection: Genomic DNA was isolated in BM cells by Qiagen DNA mine Kit. One copy Nalm-6 cell gDNA was diluted as a standard. GAG primers and Probe were synthesized by Invitrogen. Forward Sequence: GGAGCTAGAACGATTCGCAGTTA (SEQ ID NO: 5); Reverse Sequence: GGTTGTAGCTGTCCCAGTATTTGTC (SEQ ID NO: 6). Probe: ACAGCCTTCTGATGTTTCTAACAGGCCAGG (SEQ ID NO: 7)
Quantitation of hFVIII mRNA in mouse liver, spleen and lung was performed by qRT-PCR using Power SYBR Green PCR Master Mix (Applied biosystem, Warrington, UK) according to the manufacturer's instructions. Firstly, total RNA was extracted from hydrodynamically (HD) injected (pBS-HCRHPI-FVIIIA48) mouse livers, and IO treated (G-F8-LV) or naive mouse livers, spleens and lungs using TRIzol® Reagent (Life technologies, Grand Island, N.Y.). Then cDNA was generated using cDNA synthesis kit (Invitrogen, Carlsbad, Calif.). hFVIII specific primers (Forward Sequence: CCAGAGTTCCAAGCCTCCAACA (SEQ ID NO: 8); Reverse Sequence: GGAAGTCAGTCTGTGCTCCAATG (SEQ ID NO: 9)) for evaluating the FVIII transcripts and Rpl19 primers (Forward Sequence: GCATCCTCATGGAGCACAT (SEQ ID NO: 10); Reverse Sequence: CTGGTCAGCCAGGAGCTT (SEQ ID NO: 11)) for assessing the control housekeeping gene transcripts were used in qRT-PCR experiments. The hFVIII mRNA expression in HD mouse livers was used as the positive control (RQ=1) to calibrate the hFVIII mRNA expression levels in IO treated or naive mouse livers, spleens and lungs.
Results Increases of GFP-Marked Hematopoietic Lineages in Mobilization Mice after LV TransductionLV containing the ubiquitous promoter MND to drive GFP expression in mobilization mice model was used to evaluate the transduction efficiency via IV delivery of lentivirus. The mobilization drugs (G-CSF and AMD3100; see
The transduction status in BM was also analyzed after MND-GFP-LVs injection in Mobilization group and non-mobilization group. The GFP MFI (mean fluorescence intensity) of total mononuclear cells in BM displays a significant difference between mobilization group and non-mobilization group 10 weeks post LVs delivery (
GFP expression in HSCs of BM can be detected in both groups by flow cytometer after 10 weeks. MFI of the GFP+ signal in the mobilization group is 5 times higher than that in the non-mobilization group (
As in this case of LVs transducing HSCs successfully by systemic delivery under the condition of mobilization. Gp1bα promoter would drive GFP expression in megakaryocytes and storage Platelets. To evaluate the possibility, G-GFP-LVs was injected in mobilization and non-mobilization group via IV. The peak of GFP expression in platelets was at the 2nd week after G-GFP-LVs injection. Sustained GFP expression in platelets was observed in about 0.2% platelets in mobilization group at 10 weeks (
Ubiquitous expression of E-F8-LV vectors via IV injection induce formation of FVIII antibodies in vivo
To demonstrate the therapeutic benefits of this novel strategy of LVs infusion, we treated HemA mice by IV injection of the EF-hFVIII-LVs. In both mobilization group and non-mobilization group, the circulatory FVIII activity in plasma was detected by APTT assay after 2 weeks E-FVIII-LVs treatment (
FVIII protein can be stored in platelets after G-FVIII-LV transduction in vivo
The approach of G-FVIII-LVs treating HemA emerged since the G promoter can drive GFP expression and stocked in platelets. Delivery of G-FVIII-LVs for the expression of FVIII in platelets specifically has the potential to escape immune response against FVIII. To test this possibility, G-FVIII-LVs was delivered via IV; there were 1.5-4% platelets containing FVIII in the mobilization group for 3 months experimental period (
To determine if expression of FVIII in platelets could functionally compensate Hemophilia, the ROTEM® (rotational thermoelectrometry) assay was employed; this is a clotting assay to evaluate the blood clotting function by detecting the clotting time (CT), clot formation time (CFT), max clotting firmness (MCF) and a angle. The mice transduced by G-FVIII-LVs were subjected to ROTEM® assay after 6 months gene therapy. ROTEM® results confirmed a faster clotting time, shorter clotting formation time, and bigger a angle in the mobilization group compared with those in HemA mice. There were significant differences between mobilization and non-mobilization groups on a angle and MCF parameters. In the non-mobilization group, the blood clotting function did not show significant improvements, when compared with that in HemA mice (
Discussion
To obtain long-term FVIII expression after single treatment, several groups have explored different approaches of gene therapy techniques. At least four gene therapy methods are in clinical trials. BMN270 and Spark 8011 are in clinical trial phase II/III; these are gene therapy-based methods using AAV vector to delivery FVIII, with HLP promoter specifically driving expression in liver for restoring FVIII in plasma (Pasi et al., Haemophilia 22:151-152, 2016). After the treatment, FVIII are expressed in liver and secreted into blood, where it may trigger immune responses to generate anti-FVIII inhibitory antibody. Additionally, there is also a risk of the AVV being cleaned by natural inhibitor in the human body (Ertl et al., Human Gene Therapy 28:328-337, 2017; Doering et al., Molecular Therapy: J Amer Soc Gene Therapy 15:1093-1099, 2007). Gene therapy ex vivo studies also have some drawbacks, even a long-term FVIII expression and correction hemophilia phenotype were observed after implantation of transduced HSCs back to recipient animal model. Thus there is an ongoing need for the herein-described intravenous delivery of lenti-vectors in HSCs mobilization mice model, which can avoid development of inhibitors, or HSCs culture in vitro.
LVs delivery via intravenous injection was performed before. In 2006, Carbonaro and his coworkers used LV vectors via intravenous injection in mouse neonatal ADA knockout mice, and detected copy number of integrated vectors in liver and lung (Carbonaro et al., Mol Ther 13(6):1110-1120, 2006). In adult mice, IV injection was performed with detection of low copy number in BM. From those studies we learned that Lentivirus delivery via IV rendered less efficiency transduction in the HSCs of BM. The reason is that Lentivectors were eliminated by macrophages or Kupffer cells in the peripheral blood (Brown et al., Blood 109(7)2797-2805, 2007), there were less chance that vectors would migrate into bone marrow to transduce HSCs. Mobilization of HSCs combining with IVs injection is a good strategy, which promote efficiency of HSCs transduction, as the location of transduction was changed from BM to peripheral blood. Those HSCs transduced by LVs carrying FVIII would go back to BM and stay in the niche of BM for further regeneration and differentiation.
Indeed, this approach increased the efficiency of LV transduction significantly. Data herein showed that MFI is 5-fold higher in mobilization group than that in the non-mobilization group after LV injection. However, when the ubiquitous promoter was used to drive the FVIII expression secreting in plasma, either mobilization or non-mobilization group displayed higher titer of FVIII inhibitor.
FVIII driven by GP1b promoter is advantageous because it is expressed in the megakaryocytes cells, and storage of the FVIII in platelets prevents it from exposure to immunity system. When a bleeding event occurs, platelets will be activated to release the FVIII and therefore join the coagulation pathway.
To demonstrate that G-FVIII-LVs also can achieve long term therapy in HemA mice, the efficacy study was tracked up to 6 months in mouse models. The blood clotting functional data demonstrated that HemA phenotype was corrected partially for a long period. Low titer of FVIII inhibitor was observed in the non-mobilization group, although there was no significate difference compared with that mobilization group. In vitro experiment demonstrated that GP1b promoter can work in myeloid cell.
This study explored a novel strategy for gene therapy of HemA via delivery of FVIII-LVs specifically transduced HSCs by intravenous injection. This is a novel approach and offers several advantages over prior strategies, including: The IV delivery is straight forward and convenient. It does not need complicated ex vivo transplantation or cell culture in vitro. In addition, it achieves long-term efficacy by targeting HSCs. FVIII are stored in platelets, there are less chance to induce neutralization of FVIII by antibodies.
Example 7: A Novel Strategy of Lung-specific Gene Therapy for the Treatment of Hemophilia A via Intranasal Delivery of Lentiviral Vectors Encoding Factor VIIIRecently, emerging evidence showed that lung is a reservoir for haematopoietic progenitors/stem cells (HSCs) in addition to bone marrow (Lefrancias et al., Nature 544(7648):105-109, 2017). The new study reported that platelets can be produced from megakaryocytes located in the extravascular space inside the lung. Furthermore, data in the above Examples demonstrated that external factor VIII (FVIII) gene driven by the specific promoter GP1bα (G) could express FVIII protein in megakaryocytes for long-term by transducing HSCs in mice. FVIII protein stored in platelets can be released to facilitate clotting upon platelets activation. Thus, intranasal (IN) delivery of lentiviral vectors (LVs) targeting FVIII gene expression in megakaryocytes in the lung have the potential to correct HemA mice phenotype.
G-GFP-LVs were used to test if IN delivery can produce GFP expression in murine platelets. IN delivery of 24 μl G-GFP-LVs produced up to 0.075% positive GFP expression in platelets after 7 days. Although the efficiency was low, GFP expression could be detected by flow cytometer. There was a significant difference compared with the untreated control group.
To improve the transduction efficiency, higher titers/volume of LVs were used in mice pretreated with N-Acetyl Cysteine and Dexamethasone. After IN delivery of increased dosages of G-FVIII-LVs into HemA mice under the optimized protocol: Mice were treated with 130 mg/kg N-acetyl cysteine (NAC) via intravenous injection for 2 days and 10 mg/kg Dexamethasone via intraperitoneal injection 16 hrs before and 2 hrs after IN. Mice were administered with 40 μl G-FVIII-LVs (1×109 IFU/ml)/mouse via IN, then mice slept for 5 mins under isoflurane. FVIII expression in platelets was examined by intracellular staining and FVIII ELISA at day 7 post treatment. There were 0.23% FVIII positive platelets in circulating platelets and FVIII expression can be detected up to 0.3 mU/108 platelets.
Functional clotting formation was also tested by ROTEM®. The clotting parameters obtained from IN gene therapy mice were comparable to those of wild type mice, including Clotting Time (CT), Clotting Formation Time (CFT), a angle, and Max of Clotting formation (MCF). Moreover, significant difference in the CT values was observed between HemA mice group and IN gene therapy group (2641±1437(s) vs 587±354(s); p=0.032) after 7 days treatment. These data indicated successful transduction of megakaryocytes in the lung following IN delivery, leading to therapeutic correction of HemA.
Furthermore, FVIII positive platelets staining persisted at 0.23% from day 7 to day 30 post treatment. The clotting parameters tested by ROTEM® after day 30 treatment were not significantly different compared with the data after day 7 treatment, indicating potential transduction of hematopoietic stems cells by LVs. In addition, formation of anti-FVIII inhibitors was not detected by Bethesda assay.
These data showed that FVIII expressing platelets were successfully produced and were able to partially correct HemA phenotype in mice via IN delivery of G-FVIII-LVs into the lung. A benefit of FVIII expression and storage in platelet α-granules is to protect FVIII from naturalizing antibodies and to decrease the possibility of inducing inhibitory antibodies. IN delivery of LVs encoding FVIII gene targeting lung is a promising option for therapeutic treatment of hemophilia patients.
Example 8: A Novel Strategy of Lung-specific Gene Therapy for the Treatment of Hemophilia A via Intranasal Delivery of Lentiviral Vectors Encoding Factor VIIIIn this example, an embodiment of the herein described treatment method is demonstrated, using intranasal delivery of LVs to treat hemophilia A mice .
Hemophilia A (HemA) disease is a deficiency of functional plasma factor VIII (FVIII). Recently, emerging evidence showed that lung is a reservoir for haematopoietic progenitors/stem cells (HSCs) in addition to bone marrow. The new study reported that platelets can be produced from megakaryocytes located in the extravascular space inside the lung.
We utilized lentiviral vectors (LVs) driven by a Gp1bα promoter to transduce lung via intranasal delivery (IN) to generate platelet-specific FVIII gene expression for the treatment of Hemophilia A.
The vectors used in this study are provided in
Results:
G-GFP expression in platelets by G-GFP-LVs transduction via intranasal delivery is illustrated in
Expression of FVIII in platelets by G-FVIII-LV via intranasal delivery is illustrated in
Lung HSCs transduction by M-GFP-LVs is illustrated in
Conclusions: Transducing megakaryocytes in lung with lentiviral vectors via intranasal delivery achieved instant gene expression. NAC and Dex treatment helped LVs transduction efficiency. M-GFP-LVs can transduce haematopoietic stem cells in lung via intranasal delivery. Transducing haematopoietic stem cells in lung by G-FVIII-LV via intranasal delivery to achieve long-term partially cure of HemA.
As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient, or component. As used herein, the transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient, or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients, or components and to those that do not materially affect the embodiment. As used herein, a material effect would cause a statistically-significant reduction in transient expression of a therapeutic protein within 7 days following administration of a disclosed nanocarrier to a subject.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the herein-cited references and printed publications are individually incorporated herein by reference for their particular cited teachings.
It is to be understood that the embodiments disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.
The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).
Claims
1. A method of treating a subject in need of plasma Factor VIII, comprising:
- administering to the subject a therapeutically effective amount of an inflammation suppressor;
- administering to the subject a therapeutically effective amount of a CD8+ T cell depleting agent; and
- administering to the subject a therapeutically effective amount of a composition comprising a lentiviral vector comprising an optimized FVIII expression cassette expressibly linked to a megakaryocyte-specific promoter, wherein administration is via: intraosseous (IO) infusion; intravenous (IV) delivery; or intranasal (IN) or inhaled delivery.
2. The method of claim 1, wherein the inflammation suppressor comprises dexamethasone (Dex).
3. The method of claim 2, wherein the dexamethasone is administered in doses of 100 mg/kg at −24 h before, −4 h before, 4 h after, and 24 h after administration of the LV.
4. The method of claim 1, wherein the CD8+ T cell depleting agent comprises an anti-CD8 antibody.
5. The method of claim 4, wherein the CD8+ T cell depleting agent is an anti-CD8α mAb administered in doses of 4 mg/kg at −1 day before, 4 days after, and 11 days after delivery of the LV.
6. The method of claim 1, wherein the megakaryocyte-specific promoter is a GP1b-alpha promoter.
7. The method of claim 1, which method does not comprise pre-conditioning or myeloablative treatment of the subject.
8. The method of claim 1, wherein the IO infusion is carried out at a rate of 2 μL/min to 15 μL/min, 5 μL/min to 12 μL/min, or at no more than 10 μL/min.
9. The method of claim 1, wherein the IO infusion is carried out at a rate of 0.01 mL/min to 0.5 mL/min, 0.05 mL/min to 0.3 mL/min, 0.1 mL/min to 0.25 mL/min, or at no more than 0.4 mL/min.
10. The method of claim 1, wherein the IO infusion is carried out over a period of no more than 45 minutes.
11. The method of claim 1, wherein the optimized FVIII expression cassette comprises hF8X10 K12 (SEQ ID NO: 1) or hF8/N6K12RH (SEQ ID NO: 2).
12. The method of claim 1, wherein the lentiviral vector comprises SEQ ID NO: 3 or SEQ ID NO: 4, or a functional variant thereof.
13. The method of any one of the previous claims, which is a method for treating a hemostasis related disorder in the subject, and the subject is in need of such treatment.
14. The method of any one of the previous claims, wherein the subject has hemophilia A (hemA), von Willebrand disease, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated vascular coagulation (DIC), or over-anticoagulation treatment disorder.
15. The method of any one of the previous claims, wherein the subject is a HemA subject with preexisting anti-FVIII inhibitory antibodies.
16. A method of treating a subject in need of plasma Factor VIII, essentially as described herein.
Type: Application
Filed: Apr 26, 2019
Publication Date: Jul 29, 2021
Applicant: Seattle Children's Hospital D/B/A Seattle Children's Research Institute (Seattle, WA)
Inventors: Carol Hsing MIAO (Seattle, WA), David J. RAWLINGS (Seattle, WA), Chong LI (Seattle, WA)
Application Number: 17/051,137
