Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant.

BACKGROUND: The prevention of bleeding with adequately sustained levels of clotting factor, after a single therapeutic intervention and without the need for further medical intervention, represents an important goal in the treatment of hemophilia. METHODS: We infused a single-stranded adeno-associated viral (AAV) vector consisting of a bioengineered capsid, liver-specific promoter and factor IX Padua (factor IX-R338L) transgene at a dose of 5×1011 vector genomes per kilogram of body weight in 10 men with hemophilia B who had factor IX coagulant activity of 2% or less of the normal value. Laboratory values, bleeding frequency, and consumption of factor IX concentrate were prospectively evaluated after vector infusion and were compared with baseline values. RESULTS: No serious adverse events occurred during or after vector infusion. Vector-derived factor IX coagulant activity was sustained in all the participants, with a mean (±SD) steady-state factor IX coagulant activity of 33.7±18.5% (range, 14 to 81). On cumulative follow-up of 492 weeks among all the participants (range of follow-up in individual participants, 28 to 78 weeks), the annualized bleeding rate was significantly reduced (mean rate, 11.1 events per year [range, 0 to 48] before vector administration vs. 0.4 events per year [range, 0 to 4] after administration; P=0.02), as was factor use (mean dose, 2908 IU per kilogram [range, 0 to 8090] before vector administration vs. 49.3 IU per kilogram [range, 0 to 376] after administration; P=0.004). A total of 8 of 10 participants did not use factor, and 9 of 10 did not have bleeds after vector administration. An asymptomatic increase in liver-enzyme levels developed in 2 participants and resolved with short-term prednisone treatment. One participant, who had substantial, advanced arthropathy at baseline, administered factor for bleeding but overall used 91% less factor than before vector infusion. CONCLUSIONS: We found sustained therapeutic expression of factor IX coagulant activity after gene transfer in 10 participants with hemophilia who received the same vector dose. Transgene-derived factor IX coagulant activity enabled the termination of baseline prophylaxis and the near elimination of bleeding and factor use. (Funded by Spark Therapeutics and Pfizer; ClinicalTrials.gov number, NCT02484092 .).

Preclinical evaluation of an anti-HCV miRNA cluster for treatment of HCV infection.

We developed a strategy to treat hepatitis C virus (HCV) infection by replacing five endogenous microRNA (miRNA) sequences of a natural miRNA cluster (miR-17-92) with sequences that are complementary to the HCV genome. This miRNA cluster (HCV-miR-Cluster 5) is delivered to cells using adeno-associated virus (AAV) vectors and the miRNAs are expressed in the liver, the site of HCV replication and assembly. AAV-HCV-miR-Cluster 5 inhibited bona fide HCV replication in vitro by up to 95% within 2 days, and the spread of HCV to uninfected cells was prevented by continuous expression of the anti-HCV miRNAs. Furthermore, the number of cells harboring HCV RNA replicons decreased dramatically by sustained expression of the anti-HCV miRNAs, suggesting that the vector is capable of curing cells of HCV. Delivery of AAV-HCV-miR-Cluster 5 to mice resulted in efficient transfer of the miRNA gene cluster and expression of all five miRNAs in liver tissue, at levels up to 1,300 copies/cell. These levels achieved up to 98% gene silencing of cognate HCV sequences, and no liver toxicity was observed, supporting the safety of this approach. Therefore, AAV-HCV-miR-Cluster 5 represents a different paradigm for the treatment of HCV infection.

Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B.

Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.

Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microRNA cluster.

UNLABELLED: RNA interference (RNAi) is being evaluated as an alternative therapeutic strategy for hepatitis C virus (HCV) infection. The use of viral vectors encoding short hairpin RNAs (shRNAs) has been the most common strategy employed to provide sustained expression of RNAi effectors. However, overexpression and incomplete processing of shRNAs has led to saturation of the endogenous miRNA pathway, resulting in toxicity. The use of endogenous microRNAs (miRNAs) as scaffolds for short interfering (siRNAs) may avoid these problems, and miRNA clusters can be engineered to express multiple RNAi effectors, a feature that may prevent RNAi-resistant HCV mutant generation. We exploited the endogenous miRNA-17-92 cluster to generate a polycistronic primary miRNA that is processed into five mature miRNAs that target different regions of the HCV genome. All five anti-HCV miRNAs were active, achieving up to 97% inhibition of Renilla luciferase (RLuc) HCV reporter plasmids. Self-complementary recombinant adeno-associated virus (scAAV) vectors were chosen for therapeutic delivery of the miRNA cluster. Expression of the miRNAs from scAAV inhibited the replication of cell culture-propagated HCV (HCVcc) by 98%, and resulted in up to 93% gene silencing of RLuc-HCV reporter plasmids in mouse liver. No hepatocellular toxicity was observed at scAAV doses as high as 5 × 10(11) vector genomes per mouse, a dose that is approximately five-fold higher than doses of scAAV-shRNA vectors that others have shown previously to be toxic in mouse liver. CONCLUSION: We have demonstrated that exogenous anti-HCV miRNAs induce gene silencing, and when expressed from scAAV vectors inhibit the replication of HCVcc without inducing toxicity. The combination of an AAV vector delivery system and exploitation of the endogenous RNAi pathway is a potentially viable alternative to current HCV treatment regimens.

Modified infusion procedures affect recombinant adeno-associated virus vector type 2 transduction in the liver.

Recombinant adeno-associated virus (rAAV) vectors have therapeutic potential for the treatment of several types of liver diseases including hepato-deficiency disorders. Most of the preclinical and clinical applications involve the use of adeno-associated vector serotype 2 (AAV-2). However, when this vector is delivered at high doses into the portal vein or hepatic artery, a relatively small number of hepatocytes are stably transduced. We elected to determine if the route of vector administration and altering the vascular delivery route within the liver influenced the relative level of transduction. First, we delivered an AAV vector expressing the human factor IX gene from a liver-specific promoter into the hepatic artery, portal vein, or general circulation of rats. Transgene expression was equal with hepatic artery and portal vein infusion, which was higher than vector administered via peripheral venous infusion. Next, we determined how localized perfusion or changing the vector dwell time affected AAV transduction in vivo. To do this, we infused an AAV vector lacking a functional expression and quantified transduction by quantifying the number of double-stranded vector DNA genomes. By increasing vector dwell time in the liver to 5 min, vector transduction was enhanced approximately 4- to 5- fold. To establish if gene transduction could be restricted to a specific anatomic location in the liver, we delivered vector into specific liver lobes by clamping the venous inflow to the middle and left liver lobes (noninfused lobes) and infusing vector into the right two liver lobes through the hepatic artery followed by vector circulation between the two right lobes and general circulation for 5 min. With this selective infusion, 40 to 120 times higher vector genome was observed in the perfused lobes than the nonperfused lobes. All the procedures described in this study were performed without detectable liver injury or toxicity. In all, the present study clearly demonstrated that hepatic arterial infusion of rAAV is effective for liver-directed gene therapy and that other parameters related to blood flow can be adjusted to further optimize gene transfer.

Influence of vector dose on factor IX-specific T and B cell responses in muscle-directed gene therapy.

Intramuscular injection of an adeno-associated virus (AAV) vector has resulted in vector dose-dependent, stable expression of canine factor IX (cF.IX) in hemophilia B dogs with an F.IX missense mutation (Herzog et al., Nat. Med. 1999;5:56-63). The use of a species-specific transgene allowed us to study risks and characteristics of antibody formation against the therapeutic transgene product. We analyzed seven dogs that had been injected at a single time point at multiple intramuscular sites with varying vector doses (dose per kilogram, dose per animal, dose per site). Comparison of individual animals suggests an increased likelihood of inhibitory anti-cF.IX (inhibitor) development with increased vector doses, with dose per site showing the strongest correlation with the risk of inhibitor formation. In six of seven animals, such immune responses were either absent or transient, and therefore did not prevent sustained systemic expression of cF.IX. Transient inhibitory/neutralizing anti-cF.IX responses occurred at vector doses of 2 x 10(12)/site, whereas a 6-fold higher dose resulted in a longer lasting, higher titer inhibitor. Anti-cF.IX was efficiently blocked in an eighth animal that was injected with a high vector dose per site, but in addition received transient immune suppression. Inhibitor formation was characterized by synthesis of two IgG subclasses and in vitro proliferation of lymphocytes to cF.IX antigen, indicating a helper T cell-dependent mechanism. Anti-cF.IX formation is likely influenced by the extent of local antigen presentation and may be avoided by limited vector doses or by transient immune modulation.

AAV-mediated gene therapy for hemophilia.

Gene therapy for hemophilia has been contemplated since the coagulation Factor genes responsible for the disease were cloned 20 years ago. Multiple approaches towards the delivery of Factors VIII or IX, the defective genes in the most common forms of hemophilia, have resulted in positive results in animals, and largely equivocal results in human clinical testing. Use of vectors based on adeno-associated virus has led to robust and sustained cures in hemophilic mice and dogs, and intriguing preliminary results in small or ongoing clinical trials. As more clinical experience is gained, solving delivery issues will be of paramount importance and will lead to more clinical success. This success will permit hemophilia to be cured following a single injection of the normal gene.

Viral vector-mediated RNA interference.

RNA interference (RNAi) is a powerful gene silencing mechanism that if properly harnessed has the potential to revolutionize medical interventions. Delivery of inhibitory RNAs to target tissues needs to be safe, efficient, and for many diseases, long-lasting, in order to exploit this endogenous mechanism for therapeutic purposes. Viral vector systems, based on adeno-associated viruses and lentiviruses, are ideally suited to mediate RNAi because they can safely transduce a wide range of tissues and provide sustained levels of gene expression. There are now many examples of the use of viral vector-mediated RNAi to inhibit gene expression in animal models of disease, and in many cases proof-of-principle has been demonstrated. The efficient delivery of RNAi has also uncovered a number of concerns that raise questions regarding the clinical application of this technology, including off-target effects, innate immune responses, and alterations in the endogenous microRNA (miRNA) pathway. However, over the past several years, work has been done to address these problems and a number of solutions are now being implemented to mitigate these potential risks. With a deeper understanding of RNAi and continued progress in designing RNAi effectors, viral vector-mediated RNAi has the potential to change the way many diseases are treated.

Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice.

Long-term cures of hemophilia B have been achieved using AAV2 delivering the factor IX gene to the liver of adeno-associated virus (AAV)-naive hemophilic animals. However, the clinical success of this approach requires overcoming pre-existing AAV neutralizing antibodies prevalent in humans. To better define the inhibition of neutralizing antibodies on AAV2-mediated liver transduction, we developed an in vivo passive immunity model. SCID mice were first reconstituted to a defined neutralizing titer with pooled plasma-derived human immunoglobulin. AAV2-FIX vectors then were administered to the liver, and the transduction efficiency was measured by plasma FIX levels. Unexpectedly, AAV2 neutralizing titers lower than 1:10 were sufficient to neutralize 4 to 20 x 10(12) vg/kg of AAV2 vectors in vivo, a capacity that was underestimated by in vitro neutralizing assays. We also evaluated strategies to evade neutralization, including the use of alternative delivery routes, infusion parameters, empty capsids, and alternative AAV serotypes 6 and 8. The results indicate that low AAV2 neutralizing titers can be inhibitory to the tested human and primate AAV vectors delivered into the circulatory system. Therefore, novel nonprimate AAV vectors or compartmentalized delivery may offer more consistent therapeutic effects in the presence of pre-existing AAV neutralizing antibodies.

Inadvertent germline transmission of AAV2 vector: findings in a rabbit model correlate with those in a human clinical trial.

The risk of germline transmission of vector sequences in humans is a major safety concern, because the enrollment of subjects of reproductive age in early-phase clinical trials of gene transfer continues to increase. In a study of adult men with hemophilia B, adeno-associated virus serotype 2 (AAV2) delivered to the liver via the hepatic artery resulted in unexpected transient vector dissemination to the semen. Here we report that intravenous AAV2 injection in rabbits proved a useful model to assess biologic parameters of vector dissemination to the semen. Detectable vector sequences in semen disappeared in a dose-dependent and time-dependent fashion. AAV infectious particles were present only as long as day 4 after injection and were undetectable thereafter. The kinetics of vector clearance was faster in the semen fractions enriched for motile sperm than in the total semen. In addition, increased frequency of semen sampling accelerated the clearance of vector sequences from semen. Long-term follow-up, spanning hundreds of spermatogenesis cycles, showed that there was no recurrence of detectable vector sequences in semen, thus reducing the probability of inadvertent transduction of early spermatogonia not committed to differentiation at the time of vector injection. We conclude that AAV2 presents minimal germline transmission risk for humans.

Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy.

In a clinical study of recombinant adeno-associated virus-2 expressing human factor IX (AAV2-FIX), we detected 2 impediments to long-term gene transfer. First, preexisting anti-AAV neutralizing antibodies (NABs) prevent vector from reaching the target tissue, and second, CD8(+) T-cell responses to hepatocyte-cell surface displayed AAV-capsid-terminated FIX expression after several weeks. Because the vector is incapable of synthesizing viral proteins, a short course of immunosuppression, until AAV capsid is cleared from the transduced cells, may mitigate the host T-cell response, allowing long-term expression of FIX. To evaluate coad-ministration of immunosuppression, we studied AAV8 vector infusion in rhesus macaques, natural hosts for AAV8. We administered AAV8-FIX in 16 macaques via the hepatic artery and assessed the effects of (1) preexisting anti-AAV8 NABs, (2) a standard T-cell immunosuppressive regimen, and (3) efficacy and safety of AAV8-FIX. We found that low titers (1:5) of preexisting NABs abrogate transduction, whereas animals with undetectable NABs are safely and effectively transduced by AAV8-FIX. Coadministration of mycophenolate mofetil and tacrolimus with vector does not induce toxicity and does not impair AAV transduction or FIX synthesis. These findings enable a clinical study to assess the effects of immunomodulation on long-term FIX expression in patients with hemophilia B.

Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs.

Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies.

Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves long-term correction of hemophilia B in a large animal model.

In earlier work, we showed that adeno-associated virus-mediated delivery of a Factor IX gene to skeletal muscle by direct intramuscular injection resulted in therapeutic levels of circulating Factor IX in mice. However, achievement of target doses in humans proved impractical because of the large number of injections required. We used a novel intravascular delivery technique to achieve successful transduction of extensive areas of skeletal muscle in a large animal with hemophilia. We provide here the first report of long-term (> 3 years, with observation ongoing), robust Factor IX expression (circulating levels of 4%-14%) by muscle-directed gene transfer in a large animal, resulting in essentially complete correction of the bleeding disorder in hemophilic dogs. The results of this translational study establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B. These findings also have immediate relevance for gene transfer in patients with muscular dystrophy.

Preclinical gene therapy studies for hemophilia using adeno-associated virus (AAV) vectors.

Gene therapy offers a potential cure for hemophilia and several gene transfer vectors have been evaluated for their ability to treat this disease. This article reviews the studies that have been performed to evaluate the ability of recombinant adeno-associated virus (AAV) vectors to achieve safely the sustained expression of clotting factors following intramuscular, intravenous, and intrahepatic delivery to several animal models. These routes of administration are all effective in providing sustained and therapeutic levels of factor IX (FIX), although the levels vary. Intrahepatic delivery is more efficacious than intravenous administration, which is superior to intramuscular delivery. The recent development of efficient factor VIII (FVIII) expression cassettes has made AAV-based gene therapy for hemophilia A also within reach. Although no acute toxicity has been observed with any route of administration, an increased risk of antibody formation against FIX has been noted following intramuscular delivery. Biodistribution studies concluded that the vector disseminates to most tissues in a dose-dependent and time-dependent manner, but the majority of the vector resides in the targeted tissue. In addition, the risk of germline transmission has been shown to be low or absent. The relatively recent isolation of new AAV serotypes has resulted in the identification of vectors that have enhanced tropism for certain tissues. This combined with the potential of these new vectors to evade the immune response to AAV2, makes them attractive candidates for gene therapy. Although much progress has been made using AAV to treat hemophilia, there are several outstanding issues that need to be addressed. Delivery of AAV to large animals has not been reproducible, which could be due to nonoptimized delivery and/or immune responses to the vector or transgene product. In addition, a complete understanding of the biology of these vectors is required to assess their long-term safety. Solving these issues will lead to the development of a successful gene therapy product.

Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1.

Adeno-associated viral (AAV) vectors (serotype 2) efficiently transduce skeletal muscle, and have been used as gene delivery vehicles for hemophilia B and for muscular dystrophies in experimental animals and humans. Recent reports suggest that AAV vectors based on serotypes 1, 5, and 7 transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2-fold to 1000-fold. We sought to determine whether this increased efficacy could be observed in species other than mice. In immunodeficient mice we saw 10- to 20-fold higher levels of human factor IX (hF.IX) expression at a range of doses, and in hemophilic dogs we observed approximately 50-fold higher levels of expression. The increase in transgene expression was due partly to higher gene copy number and a larger number of cells transduced at each injection site. In all immunocompetent animals injected with AAV-1, inhibitory antibodies to F.IX developed, but in immunocompetent mice treated with high doses of vector, inhibitory antibodies eventually disappeared. These studies emphasize that the increased efficacy of AAV-1 vectors carries a risk of inhibitor formation, and that further studies will be required to define doses and treatment regimens that result in tolerance rather than immunity to F.IX.

Phenotypic correction of a mouse model of hemophilia A using AAV2 vectors encoding the heavy and light chains of FVIII.

Using separate adeno-associated viral 2 (AAV2) vectors to deliver the heavy and light chains of factor VIII (FVIII) we have overcome the packaging limitations of AAV, achieving phenotypic correction of hemophilia A in mice. AAV vectors were constructed that use a liver-specific promoter and the cDNA sequences of either the human or canine heavy and light chains of FVIII. After intraportal vein injection of these vectors in hemophilia-A mice, therapeutic to superphysiologic levels of active FVIII were achieved in plasma in a dose-dependent manner. Phenotypic correction of the bleeding diathesis was demonstrated by survival of all treated mice after tail clipping. Biochemical analysis demonstrated lower levels of heavy-chain (25- to 100-fold) compared with light-chain protein in the plasma of treated animals. Differences in gene transfer and transcription did not account for the differences in protein expression. We hypothesize that improvements in FVIII activity could be achieved by improvements in FVIII heavy-chain expression. This work demonstrates that cotransduction of liver with AAV vectors expressing the heavy and light chains of FVIII corrects hemophilia A in vivo, providing an alternative approach to the use of a single vector. This strategy may potentially be useful for other large therapeutic proteins that contain functionally distinct domains.

Sustained phenotypic correction of canine hemophilia A using an adeno-associated viral vector.

Gene therapy for hemophilia A requires efficient delivery of the factor VIII gene and sustained protein expression at circulating levels of at least 1% to 2% of normal. Adeno-associated viral type 2 (AAV2) vectors have a number of advantages over other viral vectors, including an excellent safety profile and persistent gene expression. However, a major disadvantage is their small packaging capacity, which has hampered their use in treating diseases such as hemophilia A, cystic fibrosis, and muscular dystrophy, which are caused by mutations in large genes. Here we demonstrate that this can be overcome by using small regulatory elements to drive expression of a B-domain-deleted form of FVIII. The use of this vector for hepatic gene transfer in a canine model of hemophilia A resulted in the sustained (> 14 months) expression of biologically active FVIII. FVIII activity levels of 2% to 4% were achieved. These levels correlated with a partial correction in the whole-blood clotting time and cuticle bleeding time. In addition, immunoprecipitation analysis demonstrated the expression of canine FVIII of the predicted size in the plasma of injected animals. These data support the use of AAV2 vectors in human clinical trials to treat hemophilia A patients.

AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B.

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (F.IX). Previously, we established an experimental basis for gene transfer as a method of treating the disease in mice and hemophilic dogs through intramuscular injection of a recombinant adeno-associated viral (rAAV) vector expressing F.IX. In this study we investigated the safety of this approach in patients with hemophilia B. In an open-label dose-escalation study, adult men with severe hemophilia B (F.IX < 1%) due to a missense mutation were injected at multiple intramuscular sites with an rAAV vector. At doses ranging from 2 x 10(11) vector genomes (vg)/kg to 1.8 x 10(12) vg/kg, there was no evidence of local or systemic toxicity up to 40 months after injection. Muscle biopsies of injection sites performed 2 to 10 months after vector administration confirmed gene transfer as evidenced by Southern blot and transgene expression as evidenced by immunohistochemical staining. Pre-existing high-titer antibodies to AAV did not prevent gene transfer or expression. Despite strong evidence for gene transfer and expression, circulating levels of F.IX were in all cases less than 2% and most were less than 1%. Although more extensive transduction of muscle fibers will be required to develop a therapy that reliably raises circulating levels to more than 1% in all subjects, these results of the first parenteral administration of rAAV demonstrate that administration of AAV vector by the intramuscular route is safe at the doses tested and effects gene transfer and expression in humans in a manner similar to that seen in animals.