Gene Therapy for Hemophilia

Abstract

The X-linked bleeding disorder hemophilia causes frequent and exaggerated bleeding that can be life-threatening if untreated. Conventional therapy requires frequent intravenous infusions of the missing coagulation protein (factor VIII [FVIII] for hemophilia A and factor IX [FIX] for hemophilia B). However, a lasting cure through gene therapy has long been sought. After a series of successes in small and large animal models, this goal has finally been achieved in humans by in vivo gene transfer to the liver using adeno-associated viral (AAV) vectors. In fact, multiple recent clinical trials have shown therapeutic, and in some cases curative, expression. At the same time, cellular immune responses against the virus have emerged as an obstacle in humans, potentially resulting in loss of expression. Transient immune suppression protocols have been developed to blunt these responses. Here, we provide an overview of the clinical development of AAV gene transfer for hemophilia, as well as an outlook on future directions.

Several successful studies of AAV-mediated, liver-targeted transfer of genes encoding clotting factors FIX and FVIII are ongoing for patients with severe hemophilia B and A, respectively. The early results of these studies and the critical questions that remain to be answered are discussed in this review.

Main Text

Hemophilia is an X-linked bleeding disorder that occurs approximately in 1 of 5,000 male births worldwide. It is caused by mutations in blood clothing factor VIII (FVIII, hemophilia A) or factor IX (FIX, hemophilia B).1, 2 Both proteins are normally produced in the liver (FIX [factor IX] by hepatocytes and FVIII by endothelial cells) and secreted into the blood circulation. FIX is a vitamin K-dependent serine protease that plays a vital role in the coagulation cascade. Its cofactor, FVIII, is critical for FIX enzymatic activity. Therefore, mutations in either protein may lead to lack of coagulation activity, resulting in frequent spontaneous bleeds in patients with activities of <1% of normal. If left untreated, bleeding may become fatal.³ Both hemophilia A and B can be treated with recombinant factor replacement with significant improvement in morbidity and mortality.³ However, such treatment is extremely costly and is often still marred by clinical complications, including bleeding, particularly bleeding into the joints despite factor replacement. The need for frequent administration of recombinant factor concentrates infused two to three times per week in order to maintain minimal therapeutic levels is demanding and highly invasive. Some improvement in outcome has been achieved in highly developed countries, but the majority of subjects throughout the world lack resources for optimal treatment.1, 2, 3, 4 These considerations prompted efforts to develop novel approaches for treatment of hemophilia using gene therapy, which has the potential for lasting treatment and even curing of the disease.

Our early gene therapy studies focused on the treatment of hemophilia B as work had progressed most rapidly for the disorder. Ongoing efforts have also accelerated with respect to the development of vectors suitable for the treatment of hemophilia A, as will be discussed in detail in this work. The work in my laboratory dedicated to the development of gene therapy for hemophilia, based on gene transfer with recombinant adeno-associated virus (rAAV), began after Dr. Amit C. Nathwani arrived from the University College London to work as a post-doctoral fellow in my lab (July 1997). Dr. Andrew Davidoff, a fully trained pediatric surgeon, was recruited to the project because of the belief that a portal-vein injection in mice was needed to ensure liver transduction. This turned out not to be true, as we have subsequently found equivalent transduction following portal versus peripheral vein infusion. They directed all of the preclinical studies and our clinical trial.

rAAV vectors have been derived from the many serotypes of AAV that exist in nature.5, 6 Serotype rAAV2 was used most extensively in early studies because had been the first serotype which was characterized. AAV viruses are thought to be nonpathogenic. The AAV genome is single stranded DNA and 7 kb in length. It includes inverted terminal repeats (ITRs) of approximately 145 base pairs. The ITRs are the only elements that must be retained in the vector genome to allow its packaging. AAV viruses depend on a helper virus for replication, usually adenovirus. Production of viral vector occurs in packaging cells that have been transfected with a multi-plasmid system in which a number of adeno-viral proteins are expressed as well as the AAV rep and capsid proteins.7, 8 Alternatively, the vector is produced using herpes virus helper or by infection of insect cells with baculovirus.7, 9

Initial studies attempting to exploit rAAV vectors for treatment of hemophilia B relied on the use of these single-stranded rAAV2 vectors. Two clinical trials have been completed, with limited results. The initial trial focused on intramuscular injection of rAAV encoding FIX in a classic dose-escalation study.10, 11, 12 Minimal expression was observed in only one of eight participants in this trial and FIX expression was at a very low level. The next trial focused on liver-targeted delivery.¹³ As liver is the natural site for FIX synthesis, this seemed like an appropriate target. Seven patients with severe hemophilia B received an rAAV2 vector via hepatic artery injection. Initial expression was observed in those trial participants who received the highest dose, but expression was lost as cytotoxic T lymphocytes appeared in the blood. They were judged to have destroyed the FIX-expressing hepatocytes.13, 14, 15

Many studies had resulted in the identification of multiple AAV serotypes.16, 17 Serotype AAV8 was particularly intriguing, in that it does not cross-react with antibodies directed against other AAV serotypes. Because AAV8 rarely infects humans, the incidence of neutralizing antibodies is relatively low, with only approximately 20% of individuals having neutralizing antibodies. Recent studies, however, suggest a significantly higher incidence of anti-AAV8 antibodies in humans.¹⁸ Furthermore, relatively low titers of the antibodies abrogate liver transduction. Two studies have shown that transient immunosuppression may facilitate transduction of the liver in vivo in animal models.19, 20

We choose serotype 8 for our studies because of its efficacy in transducing hepatocytes from peripheral vein.⁵ Another favorable factor for the AAV8 serotype is that the genome is rapidly uncoated, allowing for prompt expression.²¹ A technical advance that we incorporated into our vector design was the use of a self-complementary vector.22, 23 Self-complementary AAV vectors (scAAV) have each strand of the FIX-coding genome in an inverted orientation, between which there is an ITR which has been mutated to prevent its digestion. Self-complementary genomes quickly self-anneal when cells are transduced, enhancing the rate of gene expression as well as the level of gene expression achieved.²² We designed our vector to have a FIX expression cassette that was small enough to accommodate the self-complementary genome by modifying transcriptional control elements (enhancer, promoter, and polyadenylation site) to minimize size and therefore to allow efficient packaging of the self-complementary genome.²⁴ The FIX coding sequences were codon optimized by synthesis with the most frequently used codons in natural cellular transcripts. We observed a 20-fold improvement in FIX expression in mice compared to ssAAV vectors. Expression of FIX was very high despite the use of much lower vector doses than had been applied in earlier studies with ssAAV vectors.

Extensive preclinical studies were performed, initially in mice.²⁵ Vector was given by tail vein administration, as early studies had indicated equivalent liver uptake by peripheral injection compared to portal vein injection.²⁶ scAAV2/8 vector particles were found to be much more efficient at transducing hepatocytes than ssrAAV2/8. A dose-dependent increase in FIX was observed in mice, with animals receiving the highest dose achieving 100% of the FIX levels that are found in normal human plasma. Preclinical studies were extended to evaluate transduction of non-human primate hepatocytes.²⁵ This was achieved very successfully with a peripheral vein infusion. The vector genome was found predominantly in the liver, with lesser amounts in the spleen. Expression of the vector-encoded transcript was found exclusively in the liver as predicted based on the construction of the liver-specific enhancer promoter combination that was used. Noteworthy is the fact that no toxicity was observed in any of the animals that received vector as part of the preclinical studies. Despite the superiority of AAV8 in mice, the two serotypes seemed more equivalent in non-human primates and in the clinical trials to date.

The initial clinical trial was designed as a phase I/II dose-escalation study with the classic design.27, 28 The initial dose was 2 × 10¹¹ vector genomes (vg)/kg, with an intermediate dose of 6 × 10¹¹ vg/kg and a higher dose of 2 × 10¹² vg/kg. Overall, 12 subjects have participated in this trial. This report provides detailed results on the first 10, each of which have been followed 3 years or more after the single vector infusion. All 10 participants have had measurable levels of FIX. The two patients treated most recently also have stable production of hFIX. Among the initial six patients who received the highest dose, the average FIX level was 5.1% ± 1.7%, with each having significant production of ≥ 2%. FIX expression resulted in a dramatic reduction in requirements for FIX infusions. Several of the patients had a mild elevation in transaminase levels. After observing this phenomenon in the first high-dose patient, we resolved to begin prednisolone as soon as there was a 50% or greater increment in transaminases above the baseline values even if the values remained within the normal range. With this treatment approach, the transaminase elevations resolved over a period of a week or two and steroids could be withdrawn after 4 weeks with no recurrence of the transaminitis. Fortunately, the prompt treatment permitted maintenance of the FIX levels in the individual participants.

Several other pharmaceutical companies have recently used the AAV vector pseudotyped with a variety of capsid proteins to deliver either wild-type F9 cDNA or one containing the gain-of-function mutation known as Padua (R338L).29, 30, 31, 32 UniQure and Dimension have ongoing studies with the wild-type factor sequence, whereas Baxalta and Spark have studies entail the use F9 cDNA that incorporates the Padua mutation, which leads to an 8-fold enhancement of expression of FIX. The results of these trials, which involve over 30 patients, remain largely unpublished, but they support our initial observations.³³ The majority of these studies confirm the efficiency and durability of AAV-mediated gene transfer following systemic administration of the vector. There have been no reports of serious adverse events in any of the trials. An asymptomatic rise in liver enzymes is often associated with a decline in FIX levels has been observed in most of these studies usually between 4 and 10 weeks after gene transfer.³⁴ Treatment with corticosteroid appears to be effective at limiting the hepatocellular toxicity as well as preserving expression of transgenic FIX, especially when commenced early in some of the affected patients. The best emerging data so far is from a study sponsored by Spark Therapeutic using a novel engineered AAV capsid and a codon-optimized, and a gain-of-function Padua variant. Data presented thus far show that the low dose (5 × 10¹¹ vg/kg) of their vector resulted in sustained FIX activity levels between 12% and 63% of normal following a single administration of SPK-9001 at the initial dose level studied in the trial.29, 31

Hemophilia A is a far more common disorder than hemophilia B. Accordingly, we had great interest in developing an effective rAAV vector suitable for treating hemophilia A. Development of such a vector presented several challenges.³⁵ The human factor VIII (FVIII) coding sequence is 7 kb in length, which is too large to be packaged into an AAV capsid with its limiting packaging capacity. FVIII has three functional domains. The B domain can be deleted, and in the construction of our vector, a 226-amino-acid spacer was included in place of the B domain in the construction of our vector. Amino acid triplets which function as glycosylation sites that are normally part of the B domain were included within this spacer. The resulting 5.2-kb vector was efficiently packaged after its coding sequences were codon optimized. Super physiological levels of FVIII were achieved in mice and non-human primates.³⁵ An immune response mediated by antibodies targeted to FVIII was eliminated with transient immunosuppression.

Encouraging results from our clinical trials have stimulated a great deal of interest in the application of gene therapy for the treatment of hemophilia. Using an AAV5 containing the SQ linker codon-optimized FVIII expression cassette described above, in a study sponsored by Biomarin nine subjects with severe hemophilia A have been treated at doses ranging from 6 × 10¹² to 6 × 10¹³ vg/kg. FVIII levels in the seven patients treated at a dose of 6 × 10¹³ vg/kg have consistently been within the normal range of 40%–150%36, 37 beyond 12 weeks after gene transfer, a feat which was thought impossible to achieve just a few years ago. These data were presented at the 35th Annual J. P. Morgan HealthCare Conference and show a 91% drop in the mean annualized bleeding rate and a 98% drop in prophylactic infusions. Thus, BMN 270 opens up an exciting potentially curative treatment opportunity severe hemophilia A patients. Several review articles have also been published which outline in detail the anticipated future progress.30, 33, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50

The future of gene therapy for hemophilia looks bright. Several ongoing studies are focused on evaluating various vectors for gene delivery, strategies to enhance transduction efficiency in human hepatocytes, definition of the immune and stress responses to vector administration, and the potential application of genome editing for the treatment of these disorders.51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 The possibility of using hepatic AAV gene transfer as an immune modulatory therapy to induce tolerance to FVIII and FIX is being explored.67, 68, 69 A number of critical questions remain to be answered, however. What is the best way to avoid or overcome the CD8+ T-cell response to AAV capsid? Is it routine transient immunosuppression? Or lower, but presumably still therapeutic, doses of vector? Or manipulation of the capsid protein? What is the best AAV serotype to use, both in terms of tropism for and efficiency of infection of human hepatocytes and prevalence of pre-existing neutralizing antibodies resulting from prior wild-type AAV infection? At what point do the data suggest that this approach is safe for testing in pediatric patients, the population that would benefit most from early intervention before extensive joint damage has occurred? And will transgene expression mediated by AAV, a largely non-integrating virus, be maintained for the lifetime of a child? Will repeat administration of AAV be required, which would necessitate either the use of an alternative, non-cross-reactive serotype or immunosuppression either at initial vector administration and/or subsequent dosing(s)? Or would it be better to use an integrating virus such as lentivirus or genome editing approaches for long-term expression of normal clotting factor? Finally, can the chronic synthesis and delivery of clotting factors, as is achieved with liver-targeted gene transfer approaches, be used to induce immune tolerance in patients in whom neutralizing antibodies to FVIII or FIX have developed, a particularly difficult clinical problem?

Conflicts of Interest

A.W.N. has no conflicts of interest. A.M.D. receives patent income from Uniqure for the FIX vector and a patent has been granted for the FVIII vector. A.C.N. receives income from Uniqure for the FIX vector and income from Biomarin for FVIII. He has recently licensed a new AAV-FIX expression cassette and capsid to Freeline Therapeutics and was a founder of this company, in which he holds equity.