Hemophilia Gene Therapy: The End of the Beginning?

Abstract

Extensive preclinical research over the past 30 years has culminated in the recent regulatory approval of several gene therapy products for hemophilia. Based on the efficacy and safety data in a recently conducted phase III clinical trial, Roctavian ^® (valoctocogene roxaparvovec), an adeno-associated viral (AAV5) vector expressing a B domain deleted factor VIII (FVIII) complementary DNA, was approved by the European Commission and Food and Drug Administration (FDA) for the treatment of patients with severe hemophilia A. In addition, Hemgenix ^® (etranacogene dezaparvovec) was also recently approved by the European Medicines Agency and the FDA for the treatment of patients with severe hemophilia B. This product is based on an AAV5 vector expressing a hyper-active factor IX (FIX) transgene (FIX-Padua) transgene. All AAV-based phase III clinical trials to date show a significant increase in FVIII or FIX levels in the majority of treated patients, consistent with a substantial decrease in bleeding episodes and a concomitant reduction in factor usage obviating the need for factor prophylaxis in most patients. However, significant interpatient variability remains that is not fully understood. Moreover, most patients encountered short-term asymptomatic liver inflammation that was treated by immune suppression with corticosteroids or other immune suppressants. In all phase III trials to date, FIX expression has appeared relatively more stable than FVIII, though individual patients also had prolonged FVIII expression. Whether lifelong expression of clotting factors can be realized after gene therapy requires longer follow-up studies. Further preclinical development of next-generation gene editing technologies offers new prospects for the development of a sustained cure for hemophilia, not only in adults, but ultimately in children with hemophilia too.

INTRODUCTION AND GENERAL CONSIDERATIONS

Hemophilia A and B are X-linked genetic bleeding disorders caused by a defect in the gene that encodes for clotting factor VIII (FVIII) or factor IX (FIX), respectively. The levels of residual clotting factor activity predict the severity of the bleeding disorder, which can be classified into three severity phenotypes: mild (5–40%), moderate (1% to ≤5%), and severe (<1%).

The current standard of care requires regular prophylaxis with purified FVIII or FIX protein to maintain clotting factor activity levels above 1%, thereby preventing bleeding and preserving joint health in patients. So far, extended-half-life clotting factors, by fusing with albumin, IgG-Fc region, or other domains (Fc-vWF-XTEN), and a humanized bispecific antibody to factor IXa and factor X, have decreased the frequency of infusions.^1

–4

Though these treatments have improved patient quality of life, they are not curative and serious bleeding events persist. In addition, around 20–30% of patients with severe hemophilia A and 3% of patients with hemophilia B develop neutralizing antibodies (nAbs) against the purified clotting factor, rendering the treatment ineffective.⁵ Since hemophilia is linked to a single gene defect, it has always been considered an ideally suited target disease for gene therapy. Moreover, a severe phenotype can be converted into a moderate phenotype with even a slight increase in plasma FVIII or FIX concentrations, thereby greatly improving patient quality of life.

More than two decades ago, gene therapy showcased its potential to treat and achieve long-lasting correction of hemophilia in preclinical animal models.^6

–9 These achievements established hemophilia as one of the main catalysts for the field of gene therapy. Numerous gene therapy strategies aimed at increasing the production of FVIII and FIX have been exhaustively researched over the past 30 years.

Different vector technologies have been explored, including both integrating vectors, such as retroviral and lentiviral vectors, and non-integrating vectors, in particular recombinant adeno-associated viral (AAV) vectors. Integrating vectors have been used in the context of both liver-directed in vivo and ex vivo gene therapy. One of the most attractive target cells for ex vivo gene therapy are hematopoietic stem/progenitor cells (HSPCs) allowing expression of clotting factors in differentiated progeny, such as myeloid cells or platelets.

These studies are now being explored in phase I/II clinical trials in patients with severe hemophilia A based on encouraging preclinical studies in mice and dog models.^10
–12 Long-term expression of the clotting factors is expected by virtue of the stable genomic integration of the therapeutic transgenes in bona fide HSPCs. However, one of the challenges of this strategy is the need for myeloablative conditioning to create ‘space’ in the bone marrow niche and consequently facilitate engraftment of the engineered HSPC.

In addition, there are some concerns about the potential oncogenic risks associated with random vector integration of integrating vectors, such as γ-retroviral or lentiviral vectors.^13

–17 In particular, it is well established that transduction of HSPCs with γ-retroviral vectors can result in leukemia due to activation of oncogenes by the viral promoter/enhancer elements.^13,14

However, this genotoxic risk can be reduced by modifying the vector design through the use of weaker internal promoter/enhancer elements to drive the transgene of interest and/or by using self-inactivating vector designs in which the viral long terminal repeat had been inactivated.^15,16

Most progress has been made by using AAV vectors, which recently resulted in the first gene therapy products approved by regulatory authorities. These AAV vectors are designed to encapsulate an FVIII or FIX transgene downstream of a liver-specific promoter. The transgenes are typically codon-optimized to maximize their translation. Since AAVs are prevalent in the environment, a significant proportion of the human population has been exposed, leading to a seroprevalence of nAbs against different AAV serotypes estimated to be between 30% and 60%.

In addition, individuals who have already received an AAV vector injection will also produce nAbs, usually at a log-fold higher level than those produced in response to environmental exposure.^18
–20 This prevalence of preexisting AAV nAbs can limit vector transduction and may render the treatment ineffective. An analysis found long-lasting nAbs (±15 years) against multiple AAV serotypes in a small cohort comprising the first severe hemophilia B patients who underwent systemic AAV2-hFIX vector treatment.²¹

It will be particularly challenging to effectively overcome the relatively high and sustained levels of AAV nAbs that develop following systemic AAV vector administration. Novel strategies are currently under development to eradicate or to circumvent these preexisting nAbs.²²

To date, multiple AAV-based gene therapy clinical trials for hemophilia A or B have been completed or are currently underway. Most clinical trials are limited to AAV seronegative patients with moderate-severe hemophilia A or B who are 18 years of age or older and have not yet developed nAbs (inhibitors) against FVIII or FIX. Further, the existence of active hepatitis, chronic hepatitis C infection, general liver health, and, in certain studies, HIV infection, even if it is well controlled, are additional criteria that may restrict participation in the trials.

It is beyond the scope of this review to discuss in detail all the hemophilia gene therapy trials that have been conducted over the past two decades, which have been extensively reviewed elsewhere.^23

–26 Instead, this review will focus primarily on the latest phase III clinical trials that led to the approval of the first approved gene therapy products for hemophilia A and B that build upon earlier clinical trials in patients with severe hemophilia.^21,27
–29 In addition, we will also discuss some of the future prospects for the use of gene editing to treat adult and pediatric patients with hemophilia.

PHASE III AAV GENE THERAPY CLINICAL TRIALS FOR HEMOPHILIA A

The F8 gene has a coding sequence of 7 kb and encodes FVIII, which has three A domains, one B domain, and two C domains. Due to the small packaging capacity of AAV vectors (±4.7 kb), all clinical trials for hemophilia A have used a codon-optimized, B-domain deleted FVIII variant (BDD-FVIII).^{23

–34} This deletion of the B-domain, which comprises around 44% of the F8 complementary DNA (cDNA), preserves coagulation activity and actually increases FVIII expression at the messenger RNA (mRNA) level.^35,36

This B domain contains 19 N-linked glycosylation sites. When the B-domain is partially deleted while retaining several N-linked glycosylation sites, FVIII exhibits enhanced secretion efficiency by improving the transport from the endoplasmic reticulum (ER) to the Golgi apparatus.³⁵

Although most gene therapy strategies for hemophilia A target hepatocytes, FVIII is naturally secreted by the liver sinusoidal endothelial cells (LSECs).^37
–39 This is in agreement with transgenic mice data demonstrating that targeted F8 gene disruption in endothelial cells resulted in a severe hemophilia A phenotype whereas a hepatocyte specific F8 knockout model displayed a normal phenotype.³⁸

Nonetheless, comparable FVIII mRNA concentrations have been found in purified LSECs and hepatocytes, and tissues such as the spleen, lymph nodes, and kidney^40,41 suggesting that the production of FVIII in hepatocytes is impaired at the post-transcriptional level compared with LSECs. Functional FVIII secretion relies on a lectin, mannose-binding protein 1 (LMAN1), also known as ER/Golgi intermediate compartment-53.

LMAN1 is known to form a specific cargo receptor complex with the multiple coagulation factor deficiency protein 2 to transport specific proteins from the ER to the Golgi apparatus.⁴² High FVIII expression in hepatocytes activates an ER stress response,^43,44 which explains the impaired FVIII secretion despite the presence of FVIII mRNA.

When FVIII is highly expressed, it inhibits glucose metabolism, thereby retaining FVIII in the ER where it forms amyloid-like fibrils.⁴⁵ In addition, this aggregation is initiated by a short amino acid motif (aggron) in the A1 domain of FVIII. However, interactions with the ER chaperone proteins, calnexin/calreticulin, and immunoglobulin binding protein (BiP) were found to prevent aggregation and can, in the case of BiP, reverse it.⁴⁵

One of the largest-to-date hemophilia A phase III gene therapy clinical trials is with BioMarin's valoctocogene roxaparvovec (BMN 270). It consists of an AAV5 vector expressing a hepatocyte-specific promoter upstream of a human BDD-FVIII coding sequence.^46,47 For this phase III, single-arm, open-label trial, 134 adult male patients with severe hemophilia A (FVIII levels ≤1 IU/dL) were enrolled.

These patients previously received regular prophylaxis with exogenous FVIII and had no history of anti-FVIII or anti-AAV5 antibodies.⁴⁸ All participants received a single valoctocogene roxaparvovec infusion (6 × 10¹³ vector genomes per kilogram [vg/kg]) and were monitored for 2 or more years post-infusion. FVIII activity was measured at different time points by using the chromogenic clotting assay (CCA) and the one-stage clotting assay (OCA).⁴⁸

Among 132 participants, mean chromogenic FVIII activity increased from baseline (1 IU/dL) to week 104 by a mean of 22.3 ± 29.7 IU/dL. A mean reduction of 77.0% in annualized bleeding rate (ABR) and a 98.2% reduction of annualized rate of FVIII use from baseline were evident in 132 participants at 1 and 2 years of follow-up, indicating the superiority of valoctocogene roxaparvovec over prophylaxis exogenous FVIII use.⁴⁸

Nonetheless, 17 out of 132 participants showed a 40% loss of expression and continued decrease in FVIII activity between 1- and 2-year post-infusion, mirroring the results from a 6-year follow-up in a phase I/II BMN 270 trial with seven participants.²³ An elevation of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels was also evident in 88.8% and 35.1% of all participants, respectively, between 1- and 2-year post-infusion, indicating possible liver safety issues.⁴⁸

However, participants were treated with immunosuppressants to decrease the ALT and AST levels. Valoctocogene roxaparvovec was approved in August 2022 by the European Medicines Agency (EMA) and in June 2023 by the Food and Drug Administration (FDA) (trademark Roctavian ^®).

The efficacy and safety of giroctocogene fitelparvovec (PF-07055480, previously called SB-525), a recombinant AAV2/6 vector expressing BDD-FVIII, is currently being investigated in an ongoing phase III clinical trial (AFFINE study) after obtaining the 3-year follow-up update from the phase I/II Alta study. Giroctocogene fitelparvovec contains multi-factorial modifications to the liver-specific promoter, FVIII transgene, polyadenylation signal, and vector backbone.⁴⁹

For the phase I/II Alta study, 11 adult males with severe hemophilia A were infused with giroctocogene fitelparvovec across four ascending doses: 9 × 10¹¹ (n = 2), 2 × 10¹² (n = 2), 1 × 10¹³ (n = 2), and 3 × 10¹³ vg/kg (n = 5) and followed up for nearly 3 years (data cutoff date: May 20, 2022).²⁸ Mean FVIII activity of patients injected with the high dose increased until week 24 post-infusion to 70.1% as measured with CCA and 104.8% measured by OCA.

However, this activity gradually dropped from the 1st-year post-infusion to 12.5% (CCA) and 22.9% (OCA) at 3-year post-infusion,³³ closely mirroring the phase III data on participants who received valoctocogene roxaparvovec.⁴⁸ In this high-dose cohort, the ABR dropped to 0- after 1-year post-infusion and 0.9 throughout the entire 3-year follow-up.²⁸

However, two participants encountered an overall total of 12 bleeding events. An elevation in ALT and AST levels was evidenced in 60% and 40%, respectively, of patients receiving 3 × 10¹³ vg/kg, who were treated with corticosteroids (range: 7–135 days). From this cohort, one patient encountered serious adverse effects such as hypotension and fever 6-h post-vector infusion, which fully resolved the next day. Further investigation on the safety and efficacy of giroctocogene fitelparvovec is currently being conducted in a larger cohort phase III clinical trial.

However, some discrepancies remain when looking at the long-term FVIII expression and activity. The apparent slow decline in FVIII expression in some of the patients contradicts the 10-year stable FVIII expression observed in treated canine models of severe hemophilia A.^50,51 This loss in FVIII activity present in both clinical trials with either valoctocogene roxaparvovec or giroctocogene fitelparvovec was also observed in some of the participants who received SPK-8011, also referred to as Spark200.³¹

Interestingly, some of the participants in the SPK-8011 trial showed relatively stable FVIII expression levels for up to 3-year post-infusion.³¹ Notably, SPK-8011 treated participants received 15- to 120-fold lower vector doses (0.5–2 × 10¹² vg/kg) compared with valoctocogene roxaparvovec- and high-dose giroctocogene fitelparvovec treated patients (6 × 10¹³ and 3 × 10¹³ vg/kg, respectively), suggesting that high vector doses, and thus high FVIII concentrations, might not be favorable, resulting in a gradual decline in FVIII expression over time.

Elevation in ALT and AST levels is a common treatment-related adverse effect observed in most participants in all clinical trials. These elevations are often a result of a cytotoxic immune response to the AAV capsid, where CD8⁺ T cells recognize AAV capsid peptides presented in a major histocompatibility complex I dependent pathway by transduced hepatocytes.⁵²

If most transduced hepatocytes were to be recognized by CD8⁺ T cells, and thus be eliminated, a drop in FVIII expression would be expected. This is often observed in clinical trials where this decrease is preceded by an elevation in transaminases. Although often treatable using immunomodulation with glucocorticoids, it has not always been successful in dampening AAV-mediated immune responses, as seen in the latest phase III trial with valoctocogene roxaparvovec, where aminotransferase levels lasted from several months up to even 2-year post-infusion in 29% of participants, which could also account for the undetectable FVIII activity 2-year post-infusion in 18 out of 132 participants (13.6%).^48,53

Over the past few years, a discrepancy has been observed between plasma FVIII activity measured through CCA and OCA in several hemophilia A gene therapy clinical trials. CCA only measures the generation of activated factor X (FXa), reflecting the functionality of activated FVIII (FVIIIa). In contrast, OCA, indicated by the time needed for plasma clot formation, assesses both the conversion of the factor VIII procofactor to its active cofactor state, indicated as FVIIIa, and its downstream functionality in blood clot formation.

The activity measured by OCA tends to be 1.3 to 2.0 times higher compared with CCA.⁵⁴ This discrepancy between the activity measured with the two different assays is something that needs to be kept in mind when analyzing and comparing results from different clinical trials. Despite clinical progress and the recent conditional approval of valoctocogene roxaparvovec by the EMA and FDA, key questions remain to be addressed regarding the stability of the FVIII expression and the substantial interpatient variability.

PHASE III AAV GENE THERAPY CLINICAL TRIALS FOR HEMOPHILIA B

Compared with the F8 gene, the F9 gene has a smaller coding sequence of ∼1.6 kb, which fits easily within an AAV vector. FIX is naturally expressed in hepatocytes, making them an ideal target for gene therapy. Initial clinical trials were conducted using wild-type or codon-optimized human FIX, which in most cases resulted in stable FIX expression.^26,55
–57 However, FIX activity ranged from 2% to 5% of normal, which resulted in a bare minimum phenotypic correction and in some cases failed to prevent spontaneous and trauma-induced bleeds. In addition, the high vector doses used resulted in liver inflammation and hepatoxicity.

Therefore, it was important to lower the vector dose to avoid possible liver inflammation but still obtain high therapeutic efficiency. A major breakthrough came with the discovery of the naturally occurring FIX-R338L-Padua missense mutation in thrombophilic patients.⁵⁸ This FIX variant had an 8-fold increase in specific activity compared with wild-type human FIX, thus resulting in more robust blood clot formation.

We demonstrated that liver-directed gene therapy resulted in a 5- to 10-fold higher efficacy in hemophilia B mice, whereas Finn et al. demonstrated its superiority following muscle-directed gene therapy with AAV.^59,60 We and others also showed the superiority of FIX-Padua after AAV-based liver-directed gene therapy in preclinical animal models whereas no increased immunogenic or thrombogenic risk was apparent.^61

–64 These insights paved the way for clinical trials based on this hyperfunctional FIX-Padua, which became the gold standard for hemophilia B gene therapy clinical trials.

The phase III Health Outcomes with Padua Gene; Evaluation in Hemophilia B (HOPE-B) trial with etranacogene dezaparvovec, an AAV5 vector containing codon-optimized human FIX-Padua, showed superior results with respect to ABR and safety profile.^65,66 In this study funded by uniQure and CSL Behring, 54 adult men with hemophilia B (moderately severe n = 10; severe n = 44) with FIX activity ≤2% of normal were infused with a dose of 2 × 10¹³ vg/kg and observed over ≥52 weeks for adverse effects, FIX activity, ABR and FIX prophylaxis.^65,66

In addition, health-related quality of life (HRQoL), measured by the hemophilia-specific Hem-A QoL tool, was assessed at 6- and 12-month post-infusion. Notably, out of 54 patients, 21 had pre-existing AAV5 nAbs. One subject did not receive the full dose and was excluded from the study. Another subject did not respond to the treatment due to a high neutralizing antibody titer (nAb = 3,212).

At 6-month post-infusion, a mean FIX activity of 39.0% (OCA) or 16.5% (CCA) was achieved, which was sustained until the primary efficacy endpoint of 18 months with 36.9% (OCA) or 19.7% (CCA). Recently, Coppens et al. demonstrated a mean FIX activity of 36.7% (OCA) 24-month post-infusion and reported no clinically meaningful correlation between AAV5 nAb titer and FIX activity throughout the entire trial.⁶⁷ In comparison to the lead-in period on FIX prophylaxis, participants showed a 64% reduction in ABR and ≥95% reduction in the need for FIX prophylaxis, thereby demonstrating the statistical superiority of etranacogene dezaparvovec over FIX prophylaxis.

In addition, an overall improvement of 21.5% in HRQoL was evidenced in these participants. Safety assessment demonstrated 92 treatment-related adverse effects in 37 participants. Seventy-four out of 92 (80.4%) were mild.^65,66 Eleven subjects (20%) had elevated ALT levels, most considered as mild or moderate adverse events. Nine out of 11 participants received glucocorticoid treatment over a mean of 79.8 days, after which ALT levels reduced while maintaining stable FIX expression. In addition, FIX inhibitors did not develop in any participant.⁶⁵

This clinical trial showed stable FIX expression, increased patient eligibility even in the face of low/moderate anti-AAV5 NAbs (i.e., AAV nAb inclusion) and demonstrated an overall acceptable safety profile. Recently, etranacogene dezaparvovec, designated as Hemgenix ^®, was approved by the FDA and European Union.

Another phase III clinical trial called BENEGENE-2 is currently investigating the efficacy and safety of fidanacogene elaparvovec, also known as SPK-9001 or PF-06838435. For this study sponsored by Pfizer, 45 AAV seronegative adult men with moderately severe to severe hemophilia B were injected with 5 × 10¹¹ vg/kg AAV-LK03 vector encoding human FIX-Padua under the control of the liver-specific apolipoprotein E (ApoE) enhancer/alpha1-antitrypsin (hAAT) promoter (ApoE/hAAT).

Stable FIX activity of 27.7%, 25.5%, and 25.0% was observed at 6-, 12-, and 24-month post-infusion, respectively,⁶⁸ as well as a reduction of 71% in ABR,⁶⁹ thereby meeting its primary endpoint. The safety profile was consistent with previous phase I/II results. Elevated ALT levels were reported in 26.6% and were controlled using glucocorticosteroids. Although this trial is still ongoing, the 2-year data prove stable transgene expression and safety, closely mirroring the data obtained from the HOPE-B study, though seemingly requiring much lower doses.⁷⁰

In contrast to hemophilia A gene therapy clinical trials, these two phase III studies show that FIX expression levels are relatively stable and do not decrease over time, provided transduced hepatocytes are not cleared by an immune reaction. As with most gene therapy trials, elevations in transaminases were observed that were treated by glucocorticoid treatment while maintaining stable levels of FIX. Yet, the question remains whether this expression will remain stable over the next decades.

Patient eligibility is still a factor that needs to be improved for all AAV gene therapy. Remarkably, both AAV5 seronegative and seropositive participants of the HOPE-B study demonstrated similar FIX activity, thereby showing that AAV seropositive patients, below a certain AAV nAb threshold, are still responsive to AAV gene therapy.

No participants in either study developed nAbs against FIX-Padua or had thrombotic complications, consistent with the preclinical data^62,71 suggesting that hepatocyte expression of a transgene induces transgene protein tolerance. Some assay discrepancies, as with FVIII activity assays, were observed between the chromogenic FIX clotting- and OCA, with the latter resulting in higher activity.

However, the differences observed between the two assays are not specific to the gene therapy-derived FIX-Padua but are likely due to the inherent biochemistry of the Padua protein. Although FIX activity varies, there is a correlation between assays.⁷²

GENE EDITING

FVIII or FIX transgenes delivered with AAV vectors remain predominately episomal. Hepatocytes slowly turn over in adult patients and consequently the non-integrated transgenes are expected to be diluted on cell division. This raises some uncertainty as to whether lifelong FVIII or FIX expression will be attainable when AAV vectors are employed⁷³ in the face of slow hepatocyte turnover in adults, since transgene expression is known to decline on rapid hepatocyte division in newborn or juvenile animals.^71

–76

In contrast to AAV, the use of integrating vectors ensures long-term clotting factor expression, even in the context of rapidly proliferating hepatocytes.⁷⁷ Pediatric patients are the ideal target population for gene therapy since they can be treated before the onset of joint bleeds, arthropathy, and other hemophilia-related complications. However, since hepatocytes proliferate rapidly in these pediatric patients, it is expected that AAV delivery in this specific patient group would result in transient clotting factor expression due to dilution and loss of vector genomes.^74
–76

Consequently, current AAV-based hemophilia gene therapy strategies exclude pediatric patients with hemophilia. Given their propensity for stable genomic integration, lentiviral vectors are therefore being considered as an alternative to AAV for either liver-directed gene therapy (in adults or pediatric patients)^59,78,79 or ex vivo engineering of HSPCs.^10
–12

However, there is a potential safety risk pertaining to the quasi-random integration pattern of these vectors.^13

–17 This could potentially result in the activation of oncogenes or the inactivation or tumor suppressor genes, though this risk can be significantly reduced by careful vector design.⁸⁰

Ideally, efficient targeted integration would be preferred based on the integration of the FVIII or FIX transgenes into a so-called ‘safe harbor’ locus in the genome or by editing the defective endogenous FVIII or FIX genes themselves.⁸¹ The main advantage of editing the FVIII or FIX gene themselves is that the expression pattern and cell-type specificity will closely resemble that of the native genes since it is determined to a great extent by the endogenous promoters of these FVIII or FIX genes.

Alternatively, other ‘safe harbor’ loci may be preferred (e.g., albumin) if the promoter at those loci is stronger than the FVIII or FIX promoter, which is expected to translate in higher transgene expression levels.⁸¹ Gene editing-based approaches for hemophilia treatment require the making of targeted double-strand breaks (DSBs) in the genome, and then inserting or correcting a copy of the gene at that location.

In the context of hemophilia gene therapy, these DSBs are induced by using either zinc finger nuclease (ZFN)-based strategies or CRISPR/Cas technologies. ZFNs were able to induce DSBs at the desired genomic locus when delivered directly to mouse livers and, when co-delivered with an appropriately designed gene-targeting vector, gene replacement was enabled at the ZFN-specified locus through both homology-directed and homology-independent targeted gene insertion.^82,83

Proof of concept was first established that hemophilia could be partially corrected by gene editing with ZFNs in hemophilic mice, which was sustained even in the face of induced hepatocyte proliferation due to targeted stable genomic integration of the transgene.⁸² Based on these preclinical studies, a clinical trial was subsequently conducted in patients with severe hemophilia B by ZFN-based gene editing.^84,85

In this method, two ZFN molecules delivered via AAV were designed to target the albumin locus as a safe harbor target site to allow insertion of a donor FIX cDNA, supplied by another AAV, downstream of the albumin promoter. Consequently, effective gene editing requires the presence of these three different constructs in each transduced cell.

Unfortunately, the treated hemophilia B subject was not able to decrease the use of FIX concentrate and genome editing could not be assessed. This phase I/II trial was discontinued, indicating that this gene editing strategy requires further optimization before sustained phenotypic correction might ultimately be realized.

There have been multiple reports demonstrating the potential of CRISPR/Cas for gene editing of hemophilia A or B as an alternative to ZFNs. In these cases, either the defective clotting factor gene itself was corrected in situ or the albumin locus was used as a safe harbor, co-opting the powerful albumin promoter to drive FVIII or FIX. Different types of Cas variants and gene delivery methods (AAV, adenoviral, hydrodynamic transfection) were explored in these studies.^{76,86

–94}

Typically, sustained clotting factor levels and at least partial correction of the bleeding diathesis could be achieved, even in neonates or in conditions of induced hepatocyte proliferation. FIX levels in the physiologic range or higher could be attained by using the codon-optimized FIX-Padua instead.^{71,86

–94}

Intellia therapeutics integrated a functional copy of FIX cDNA into the intron of the safe harbor albumin locus.⁹⁵ To achieve this, they employed non-viral lipid nanoparticles (LNPs) to deliver CRISPR/Cas9 mRNA and albumin guide RNA (gRNA), as well as an AAV encoding the F9 cDNA. This hybrid LNP-AAV approach allowed for precise integration into the genome, resulting in stable expression of FIX driven by the endogenous Alb promoter, and transient expression of Cas9, thereby decreasing the chance of off-target cuts.⁹⁵

The use of LNPs has several advantages: it allows for redosing, they have a large cargo capacity, and they have a low immunogenicity profile compared with AAV vectors. Targeted integration of around 50% in liver cells was obtained, resulting in 10-month stable plasma FIX concentration in adult hemophilia B mice. Similarly, normal levels of circulating FIX proteins were maintained through day 28 post-injection in non-human primates.⁹⁵

It is important to assess AAV integration patterns in the context of CRISPR/Cas-based gene editing. Although AAV vector genomes are known to reside episomally or integrate randomly into pre-existing DSBs across the genome, we and others have observed that AAV vector genomes can also integrate into specific CRISPR-induced DSBs.^96
–98

This may raise potential genotoxicity concerns due to continuous Cas9 and gRNA expression resulting from the integrated AAV vector copies encoding the CRISPR/Cas components. Breton et al. developed a next-generation sequencing assay called ITR-Seq to detect in vivo AAV integration in genome-wide DNA editing sites and will help us understand the specificity and efficacy of genome-editing nucleases in preclinical and clinical studies.⁹⁸

Though CRISPR/Cas is typically required to augment the efficiency of the gene targeting, other studies showed that targeting could also be achieved even without relying of CRISPR/Cas9 or other nucleases to inducing DSBs In particular, Barzel et al. developed a strategy referred as “GeneRide,” where a promoterless FIX gene, preceded by a 2A-peptide coding sequence and flanked by homology arms spanning the mouse Alb stop codon, is integrated by homologous recombination into the albumin gene.⁹⁹

This resulted in an on-target integration frequency of ∼0.5%, and FIX levels of up to 20% in neonates and adult hemophilia B mice.⁹⁹ This 2A-fusion integration strategy allows us to efficiently target genes and treat genetic diseases in both newborns and adults while significantly reducing the off-target effects.⁹⁹

CONCLUSIONS

The approval of the first gene therapy products for hemophilia A (BioMarin's Roctavian—valoctocogene roxaparvovec) and hemophilia B (CSL Behring/uniQure's Hemgenix—etranacogene dezaparvovec) heralds a new chapter in the treatment of patients with severe hemophilia. This represents not just an important milestone for hemophilia but also a significant step in our battle against disease and human suffering with a potentially broader impact for the field at large.

Other phase III gene therapy trials for hemophilia A or B are still ongoing and are based on different vector designs, capsids, and/or manufacturing methods. The available data from these trials are encouraging in that therapeutic FVIII or FIX levels can be attained that are consistent with a reduction of factor consumption and bleeding frequency, in accordance with the results obtained in the pivotal trials for the approved products.

In the absence of AAV standards and head-to-head comparisons of vector titer and quality, it is not straightforward to compare dose responses in these different clinical studies. This caveat notwithstanding, it would appear that in some trials much lower vector doses were required to achieve comparable clinical benefits and factor levels. This may possibly reflect differences in vector designs, capsids, and/or manufacturing methods.

Despite this progress, there are other challenges ahead, and the state of play could perhaps best be summed up by one of Sir Winston Churchill's famous quotes: “Now this is not the end. It is not even the beginning of the end. But it is, perhaps, the end of the beginning” (London, November 10, 1942). One of the main challenges that needs to be addressed is the high variability among the treated patients, resulting in a substantial variation in FVIII or FIX levels after gene therapy.

The exact reasons for these differences are not fully understood but may reflect differences at the level of vector entry, intracellular trafficking, regulation of FVIII or FIX expression itself, and/or immune mechanisms. Notably, in a previous AAV-based gene therapy trial for hemophilia B, high stable FIX expression levels were associated with a polymorphism in the interleukin-6 (IL-6) gene, though no cause-effect relationship had formally been established.¹⁰⁰

Humoral and cellular immune responses against the transgene product or the gene-modified cells can be triggered after receiving AAV gene therapy. This immune response depends on several confounding variables, including the nature of the transgene itself, the underlying mutation of the defective endogenous gene, the route of vector administration and transduced target cell, the vector dose, and the vector design.

Preclinical studies have demonstrated that untoward immune reactions against the transgene product or the transduced cells can be prevented and/or suppressed by induction of immune tolerance. Notably, induction of immune tolerance relies on the induction of transgene-specific regulatory T cells (CD4⁺CD25⁺FoxP3⁺ Treg cells) that are believed to play a role in the induction of immune tolerance after hepatic gene therapy with AAV or other vectors.^101

–104

Liver resident antigen-presenting cells, such as Kupffer cells, hepatocytes, stellate cells, and LSECs, are known to play a role in the induction of tolerance by secreting IL-10 and transforming growth factor-β, Notch signaling, presenting hepatocyte-derived antigens to Tregs, and depleting effector T cells in a Fas/FasL dependent pathway.^105

–108

To induce tolerance, Tregs must be induced and antigen-specific CD8⁺ T cells must have their programmed cell death protein-1 (PD-1) pathway activated.¹⁰¹ Notably, activation of the PD-1 pathway does not influence the activation of CD8⁺ T cells but does impact their functionality once activated. The magnitude of tolerance induction and CD8⁺ T cell-mediated clearance is further determined by the used viral vector dose.¹⁰⁹

In addition, tolerance toward the clotting factor can also be achieved, as seen in the research of Chen et al. where hemophilia A mice were injected with a highly Treg-selective mutated version of murine IL-2 before receiving gene therapy. This resulted in tolerance toward FVIII by preventing inhibitor formation, which was stably maintained for at least 6 months.¹¹⁰ In addition, tolerance can also be achieved by a combination of rapamycin and ibrutinib, which blocks the antibody-mediated complement activation toward the AAV capsid and produced clotting factor.¹¹¹ Similarly, immune modulation with anti-CD20 and rapamycin resulted in a similar disruption of FVIII inhibitor formation.¹¹²

Adaptive and innate immune mechanisms likely played a role in the vector dose-dependent transaminitis that is frequently observed in all hemophilia gene therapy trials, but more research is needed to further dissect these mechanisms. Though transient immune suppressive treatment with corticosteroids helped to stabilize factor expression in many subjects, this was not always effective, and triggered known side-effects.

Consequently, it is important to continue to develop strategies that allow for a further reduction in vector doses without compromising the overall efficacy by vector and transgene engineering. For instance, we recently tested a new bioengineered transgene (in collaboration with Dr. Blouse and colleagues), designated as CB 2679d-GT, which had improved catalytic activity, affinity for activated FVIII, and resistance to antithrombin inhibition.¹¹³

This novel transgene significantly outperformed FIX-Padua and allows further reduction in AAV vector dose while enhancing therapeutic efficacy, thus making it a potentially attractive candidate for future clinical trials.

Another unresolved issue concerns the apparently different outcomes in the hemophilia A versus B trials, where FVIII expression appears to be less stable than FIX. Although still unclear, several mechanisms have been proposed that could explain this decline in FVIII activity, including: (1) F8 gene silencing, (2) FVIII mediated unfolded protein response, (3) undetected immune response against transduced cells (against AAV capsid and/or FVIII), or (4) vector characteristics related to the vector production method.^{45,114

–118} Targeting FVIII expression at endothelial cells, which are the naturally FVIII-producing cells, may potentially overcome some of these concerns caused by FVIII over-expression in ectopic cell types such as hepatocytes.

Despite the demonstration of multiyear expression in small and large animal models and in phase III trials in hemophilia A and B patients, it is still uncertain whether AAV gene therapy will result in lifelong expression of the clotting factors. Gene editing may, therefore, offer new prospects that could ultimately result in a bona fide cure. However, given the disappointing results of the previous ZFN-based gene editing trials in hemophilia, it would be preferable to conduct preclinical studies in large animal models first using the latest advances in CRISPR technology.

CRISPR-based studies in mouse models typically rely on AAV as a vector to deliver the CRISPR/Cas components but their prolonged expression increases the risk of potential off-target events or DSB-induced side effects such as chromothripsis or p53-mediated DNA damage responses.^119,120

There is, therefore, still a need to develop efficient non-viral gene delivery approaches to introduce the CRISPR/Cas components along with the donor template to the desired target cells. Ideally, a ‘hit-and-run’ approach would be preferable during which the CRISPR/Cas components would only be present in the transfected cells during a critical window to achieve efficient gene editing, after which their presence is no longer required.

Finally, it cannot be excluded that expression of the bacterial Cas9 protein, or its derivatives and orthologues, may evoke an untoward immune response that results in the elimination of the gene-edited cells.¹²¹

Whether gene editing for hemophilia will ultimately fulfill its promise and overcome some of the challenges of conventional gene therapy in the face of “known unknowns” and “unknown unknowns” is currently far from certain and only comprehensive evidence-based preclinical studies and well-designed clinical trials will ultimately provide some of the answers.

Footnotes

AUTHOR DISCLOSURE

T.V. and M.K.C. are inventors on patents in gene therapy for hemophilia. They have received funding from Takeda, Pfizer, Bayer and Catalyst Biosciences. T.V. received speaker honoraria from Takeda, Pfizer and BioMarin. The remaining authors declare no competing interests.

FUNDING INFORMATION

This work was supported by grants from the EU Horizon 2020 grant (UPGRADE—Unlocking Precision Gene Therapy); Grant Agreement No. 825825; VUB Strategic Research Program “Groeier”; VUB Industrieel Onderzoeksfonds (Groups of Expertise in Applied Research to M.K.C. and T.V.D.).

References

Peyvandi

, Garagiola

, Young

. The past and future of haemophilia: Diagnosis, treatments, and its complications. Lancet, 2016; 388(10040):187–197.

Oldenburg

. Optimal treatment strategies for hemophilia: Achievements and limitations of current prophylactic regimens. Blood, 2015; 125(13):2038–2044.

Lillicrap

. Bispecific antibody therapy in hemophilia. N Engl J Med, 2017; 377(9):884–885.

VandenDriessche

, Tatsumi

, Shima

. Should patients with haemophilia receive gene therapy?. Lancent Haematol, 2022; 9(10):722–723.

Dimichele

. Inhibitors: Resolving diagnostic and therapeutic dilemmas. Haemophilia, 2002; 8(3):280–287.

Evens

, Chuah

, VandenDriessche

. Haemophilia gene therapy: From trailblazer to gamechanger. Haemophilia, 2018; 24:50–59.

Hasbrouck

, High

. AAV-mediated gene transfer for the treatment of hemophilia B: Problems and prospects. Gene Ther, 2008; 15:870–875.

High

. The gene therapy journey for hemophilia: Are we there yet?. Blood, 2012; 120(23):4482–4487.

Nathwani

, Davidoff

, Tuddenham

EGD

. Prospects for gene therapy of haemophilia. Haemophilia, 2004; 10:309–318.

10.

Shi

, Kuether

, Chen

, et al. Platelet gene therapy corrects the hemophilic phenotype in immunocomprised hemophilia A mice transplanted with genetically manipulated human cord blood stem cells. Blood, 2014; 123(3):395–403.

11.

Doering

, Denning

, Shields

, et al. Preclinical development of a hematopoietic stem and progenitor cell bioengineered factor VIII lentiviral vector gene therapy for hemophilia A. Hum Gene Ther, 2018; 29(10):1183–1201.

12.

, Nurden

, et al. Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A. Nat Commun, 2013; 4:2773.

13.

, Düllmann

, Schiedlmeier

, et al. Murine leukemia induced by retroviral gene marking. Science, 2002; 296:497.

14.

Hacein-Bey-Abina

, von Kalle

, Schmidt

, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med, 2003; 348:255–256.

15.

Montini

, Cesana

, Schmidt

, et al. The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy. J Clin Invest, 2009; 119(4):964–975.

16.

Cesana

, Ranzani

, Volpin

, et al. Uncovering and dissecting the genotoxicity of self-activating lentiviral vectors in vivo. Mol Ther, 2014; 22:774–785.

17.

Dull

, Zufferey

, Kelly

, et al. A third-generation lentivirus vector with a conditional packaging system. J Virol, 1998; 72:8463–8471.

18.

Calcedo

, Vandenberghe

, Gao

, et al. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis, 2009; 199(3):381–390.

19.

Boutin

, Monteilhet

, Veron

, et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: Implications for gene therapy using AAV vectors. Hum Gene Ther, 2010; 21(6):704–712.

20.

Klamroth

, Hayes

, Andreeeva

, et al. Global seroprevalence of pre-existing immunity against AAV5 and other AAV serotypes in people with hemophilia A. Hum Gene Ther, 2022; 33(7–8):32–41.

21.

George

, Ragni

, Rasko

JEJ

, et al. Long-term follow-up of the first in human intravascular delivery of AAV for gene transfer: AAV2-hFIX16 for severe hemophilia B. Mol Ther, 2020; 28:2073–2082.

22.

Weber

. Anti-AAV antibodies in AAV gene therapy: Current challenges and possible solutions. Front Immunol, 2021; 12:658399.

23.

Samelson-Jones

, George

. Adeno-associated virus gene therapy for hemophilia. Annu Rev Med, 2023; 74:231–247.

24.

Perrin

, Herwog

, Markusic

. Update on clinical gene therapy for hemophilia. Blood, 2019; 133(5):407–414.

25.

Nienhuis

, Nathwani

, Davidoff

. Gene therapy for hemophilia. Mol Ther, 2017; 25(5):1163–1167.

26.

Nathwani

, Reiss

, Tuddenham

EGD

, et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. New Engl J Med, 2014; 371(21):1994–2004.

27.

George

, Sullivan

, Giermasz

, et al. Hemophilia B gene therapy with high-specific-activity factor IX variant. N Engl J Med, 2017; 377(23):2215–2227.

28.

Rangarajan

, Walsh

, Lester

, et al. AAV5-factor VIII gene transfer in severe hemophilia A. N Engl J Med, 2017; 377:2519–2530.

29.

Pasi

, Rangarajan

, Mitchell

, et al. Multiyear follow-up of AAV5-hFVIII-SQ gene therapy for hemophilia A. N Engl J Med, 2020; 382(1):29–40.

30.

George

, Monahan

, Eyster

, et al. Multiyear factor VIII expression after AAV gene transfer for hemophilia A. N Engl J Med, 2021; 385:1961–1973.

31.

George

, Ragni

, Samelson-Jones

, et al. Spk-8011: Preliminary results from a phase 1/2 dose escalation trial of an investigational AAV-mediated gene therapy for hemophilia A. Blood, 2017; 130(Suppl. 1):604.

32.

Giermasz

, Visweshwar

, Harrington

, et al. Updated results of the Alta Study, a phase 1/2 study of giroctocogene fitelparvovec (PF-07055480/SB-525) gene therapy in adults with severe hemophilia A. Blood, 2022; 140(Suppl. 1):7776–7777.

33.

Pipe

, Hay

CRM

, Sheehan

, et al. First-in-human gene therapy study of AAVhu37 capsid vector technology in severe hemophilia A: Safety and FVIII activity results. Res Pract Thromb Haemost, 2020; 4(Suppl. 1):OC 09.4 (Abstr.)

34.

Ward

, Buckley

, Waddington

, et al. Codon optimization of human factor VIII cDNAs leads to high-level expression. Blood, 2011; 117(3):798–807.

35.

Croteau

, Wang

, Wheeler

. 2021 clinical trials update: Innovations in hemophilia therapy. Am J Hematol, 2021; 96:128–144.

36.

Miao

, Sirachainan

, Palmer

, et al. Bioengineering of coagulation factor VIII for improved secretion. Blood, 2004; 103:3412–3419.

37.

Everett

, Cleuren

ACA

, Khoriaty

, et al. Murine coagulation factor VIII is synthesized in endothelial cells. Blood, 2014; 123(24):3697–3705.

38.

Fahs

, Hille

, Shi

, et al. A conditional knockout model reveals endothelial cells as the principal and possible exclusive source of plasma factor VIII. Blood, 2014; 123(24):3706–3713.

39.

Shahani

, Covens

, Lavend'homme

, et al. Human liver sinusoidal endothelial cells but not hepatocytes contain factor VIII. J Thromb Haemost, 2013; 12(1):36–42.

40.

, Healey

, Waller

, et al. Expression of factor VIII by murine liver sinusoidal endothelial cells. J Biol Chem, 1999; 274(28):19587–19592.

41.

Wion

, Summerfield

, Tuddenham

, et al. Distribution of factor VIII mRNA and antigen in human liver and other tissues. Nature, 1985; 317(6039):726–729.

42.

Zhang

, Cunningham

, Nichols

, et al. Bleeding due to disruption of a cargo-specific ER-to-Golgi transport complex. Nat Genet, 2003; 34(2):220–225.

43.

Fong

, Handyside

, Sihn

, et al. Induction of ER Stress by an AAV5 BDD FVIII construct is dependent on the strength of the hepatic-specific promoter. Mol Ther Methods Clin Dev, 2020; 18:620–630.

44.

Lange

, Altynova

, Nguyen

, et al. Overexpression of factor VIII after AAV delivery is transiently associated with cellular stress in hemophilia A mice. Mol Ther Methods Clin Dev, 2016; 3:16064.

45.

Poothong

, Pottekat

, Siirin

, et al. Factor VIII exhibits chaperone-dependent and glucose-regulated reversible amyloid formation in the endoplasmic reticulum. Blood, 2020; 135(21):1899–1911.

46.

Ozelo

, Mahlangu

, Pasi

, et al. Valoctocogene roxaparvovec gene therapy for hemophilia A. N Engl J Med, 2022; 386:1013–1025.

47.

Bunting

, Zhang

, Xie

, et al. Gene therapy with BMN 270 results in therapeutic levels of FVIII in mice and primates and normalization of bleeding in hemophilic mice. Mol Ther, 2018; 26:694–705.

48.

Mahlangu

, Kaczmarek

, von Drygalski

, et al. Two-year outcomes of valoctocogene roxaparvovec therapy for hemophilia A. N Engl J Med, 2023; 388(8):694–705.

49.

National Hemophilia Foundation. FDA places the Pfizer/Sangamo Therapeutic phase 3 AFFINE haemophilia A gene therapy study on clinical hold. 2021. Available from: https://www.hemophilia.org/news/fda-places-the-pfizersangamo-therapeutics-phase-3-affine-haemophilia-a-gene-therapy-study-on-clinical-hold [Last accessed: June 7, 2023 ].

50.

Nguyen

, Everett

, Kafle

, et al. A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells. Nat Biotechnol, 2021; 39(1):47–55.

51.

Batty

, Mo

, Hurlbut

, et al. Long-term follow-up of liver-directed, adeno-associated vector-mediated gene therapy in the canine model of hemophilia A. Blood, 2022; 140(25):2672–2683.

52.

Mingozzi

, Maus

, Hui

, et al. CD8⁺ T-cell responses to adeno-associated virus capsid in humans. Nat Med, 2007; 13(4):419–422.

53.

Lindsey

, George

. Hemophilia A gene therapy—Some answers, more questions. N Engl J Med, 2023; 388:761–763.

54.

Rose

, Tiefenbacher

, Robinson

, et al. Activity of transgene-produced B-domain-deleted factor VIII in human plasma following AAV5 gene therapy. Blood, 2020; 136(22):2524–2534.

55.

Manno

, Pierce

, Arruda

, et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med, 2006; 12(3):342–347.

56.

Nathwani

, Tuddenham

, Rangarajan

, et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med, 2011; 365(25):2357–2365.

57.

Miesbach

, Meijer

, Coppens

, et al. Gene therapy with adeno-associated virus vector 5–human factor IX in adults with hemophilia B. Blood, 2018; 131(9):1022–1031.

58.

Simioni

, Tormene

, Tognin

, et al. X-linked thrombophilia with a mutant factor IX (factor IX Padua). N Engl J Med, 2009; 361:1671–1675.

59.

Cantore

, Nair

, Della Valle

, et al. Hyperfunctional coagulation factor IX improves the efficacy of gene therapy in hemophilic mice. Blood, 2012; 120:4517–4520.

60.

Finn

, Nichols

, Svoronos

, et al. The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy. Blood, 2012; 120:4521–4523.

61.

Nair

, Rincon

, Evens, et al. Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy. Blood, 2014; 123:3195–3199.

62.

Crudele

, Finn

, Siner

, et al. AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice. Blood, 2015; 125(10):1553–1561.

63.

Monahan

, Sun

, Gui

, et al. Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: Preclinical evaluation supporting an ongoing adeno-associated virus clinical trial. Hum Gene Ther, 2015; 26:69–81.

64.

Kao

, Yang

, Tao

, et al. Incorporation of the factor IX Padua mutation into FIX-Triple improves clotting activity in vitro and in vivo. Thromb Haemost, 2013; 110:244–256.

65.

Pipe

, Leebeek

FWG

, Recht

, et al. Gene therapy with etranacogene dezaparvovec for hemophilia B. N Engl J Med, 2023; 388:706–718.

66.

Pipe

, Leebeek

FWG

, Recht

, et al. Stable hemostatic correction and improved hemophilia-related quality of life: Final analysis from the pivotal phase 3 HOPE-B trial of etranacogene dezaparvovec . Mol Ther, 2022; 30(4)(Suppl. 1):(Abstr.)

67.

Coppens

, Pipe

, Miesbach

, et al. Adults with hemophilia B receiving etranacogene dezaparvovec in the HOPE-B phase 3 trial experiencing a stable increase in mean factor IX activity and durable haemostatic protection after 24 months' follow-up. Presented at the 16th Annual Congress of the European Association for Haemophilia and Allied Disorders (EAHAD), February 7–10, 2023.

68.

Kavakli

Efficacy and safety of Fidanacogene elaparvovec in adults with moderately severe to severe hemophilia B: The phase 3 BENEGENE-2 gene therapy trial. Presented at the 16th Annual Congress of the European Association for Haemophilia and Allied Disorders (EAHAD), February 7–10, 2023.

69.

Meisbach

Long-Term Data from Phase 3 Studies on FIX Gene Therapy. NHF: Washington D.C.; 2023 (Abstr.).

70.

Pfizer. Pfizer announces positive top-line results from phase 3 study of hemophilia B gene therapy candidate. Pfizer Press Release; 2022. Available from: https://www.pfizer.com/news/press-release/press-release-detail/pfizer-announces-positive-top-line-results-phase-3-study [Last accessed: June 14, 2023 ].

71.

Mingozzi

, Hasbrouck

, Basner-Tschakarjan

, et al. Modulation of tolerance to the transgene product in a nonhuman primate model of AAV-mediated gene transfer to liver. Blood, 2007; 110(7):2334–2341.

72.

Robinson

, George

, Carr

, et al. Factor IX assay discrepancies in the setting of liver gene therapy using a hyperfunctional variant factor IX-Padua. J Thromb Haemost, 2021; 19(5):1212–1218.

73.

Colella

, Ronzitti

, Mingozzi

. Emerging issues in AAV-mediated in vivo gene therapy. Mol Ther Methods Clin Dev, 2017; 8:87–104.

74.

Zhang

, Yates

, Murphy

, et al. Young mice administered adult doses of AAV5-hFVIII-SQ achieve therapeutic factor VIII expression into adulthood. Mol Ther Methods Clin Dev, 2022; 26:519–531.

75.

Flageul

, Aubert

, Pichard

, et al. Transient expression of genes delivered to newborn rat liver using recombinant adeno-associated virus 2/8 vectors. J Gene Med, 2009; 11:689–696.

76.

Singh

, Evens

, Nair

, et al. Efficient in vivo liver-directed gene editing using CRISPR/Cas9. Mol Ther, 2018; 26(5):1241–1254.

77.

VandenDriessche

, Vanslembrouck

, Goovaerts

, et al. Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice. Proc Natl Acad Sci USA, 1999; 96(18):10379–10384.

78.

Milani

, Canepari

, Liu

, et al. Liver-directed lentiviral gene therapy corrects hemophilia A mice and achieves normal-range factor VIII activity in non-human primates. Nat Commun, 2022; 13(1):2454.

79.

Milani

, Annoni

, Moalli

, et al. Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates. Sci Transl Med, 2019; 11(493):eaav7325.

80.

Cantore

, Ranzani

, Bartholomae

, et al. Liver-directed lentiviral gene therapy in a dog model of hemophilia B. Sci Transl Med, 2015; 7(277):277ra28.

81.

Papapetrou

and Schambach

. Gene insertion into genomic safe harbours for human gene therapy. Mol Ther, 2016; 24(4):678–684.

82.

, Haurigot

, Doyon

, et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature, 2011; 475(7355):217–221.

83.

Anguela

, Sharma

, Doyon

, et al. Robust ZFN-mediated genome editing in adult hemophilic mice. Blood, 2013; 122(19):3283–3287.

84.

Sangamo Therapeutics. A Phase I, Open-label, Ascending dose study to assess the safety and tolerability of AAV2/6 factor IX gene therapy via zinc finger nuclease (ZFN) mediated targeted integration of SB-FIX in adult subjects with severe hemophilia B. Clinicaltrials.gov; 2022. Available from: https://classic.clinicaltrials.gov/ct2/show/study/NCT02695160 [Last accessed: July 4, 2023 ].

85.

Harmatz

, Prada

, Burton

, et al. First-in-human in vivo genome editing via AAV-zinc-finger nucleases for mucopolysaccharidosis I/II and hemophilia B. Mol Ther, 2022; 30:3587–3600.

86.

Guan

, Ma

, Li

, et al. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol Med, 2016; 8(5):477–488.

87.

Ohmori

, Nagao

, Mizukami

, et al. CRISPR/Cas9-mediated genome editing via postnatal administration of AAV vector cures haemophilia B mice. Sci Rep, 2017; 7(1):4159.

88.

Huai

, Jia

, Sun

, et al. CRISPR/Cas9-mediated somatic and germline gene correction to restore hemostasis in hemophilia B mice. Hum Genet, 2017; 136(7):875–883.

89.

Wang

, Yang

, Breton

, et al. CRISPR/Cas9-mediated in vivo gene targeting corrects hemostasis in newborn and adult factor IX-knockout mice. Blood, 2019; 133:2745–2752.

90.

Chen

, Shi

, Gilam

, et al. Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human factor VIII. Sci Rep, 2019; 9(1):16838.

91.

Zhang

, Cheng

, Zhao

, et al. Curing hemophilia A by NHEJ-mediated ectopic F8 insertion in the mouse. Genome Biol, 2019; 20(1):276.

92.

Wang

, Zhong

, Li

, et al. CRISPR-Cas9-mediated in vivo gene integration at the albumin locus recovers hemostasis in neonatal and adult hemophilia B mice. Mol Ther Methods Clin Dev, 2020; 18:520–531.

93.

Luo

, Li

, Dai

, et al. CRISPR/Cas9-mediated in vivo correction in a mouse model of hemophilia A. Front Cell Dev Biol, 2021; 9:672564.

94.

Lee

, Oh

, Choi

, et al. Genome editing-mediated knock-in of therapeutic genes ameliorates the disease phenotype in a model of hemophilia. Mol Ther Nucleic Acids, 2022; 29:551–562.

95.

Hon-Ren Huang. CRISPR/Cas9-mediated targeted insertion of human F9 achieves therapeutic circulating protein levels in mice and non-human primates. Presented at the 22nd Annual Meeting of the American Society of Gene and Cell Therapy, April 29, 2019.

96.

Miller

, Petek

, Russel

. Adeno-associated virus vectors integrate at chromosome breakage sites. Nat Genet, 2004; 36:767–773.

97.

Hanlon

, Kleinstiver

, Garcia

, et al. High levels of AAV vector integration into CRISPR-induced DNA breaks. Nat Commun, 2019; 10:4439.

98.

Breton

, Clark

, Wang

, et al. ITR-Seq, a next-generation sequencing assay, identifies genome-wide DNA editing sites in vivo following adeno-associated viral vector-mediated genome editing. BMC Genomics, 2020; 21:239.

99.

Barzel

, Paulk

, Shi

, et al. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature, 2015; 517(7534):360–364.

100.

Konkle

, Walsh

, Escobar

, et al. BAX 335 hemophilia B gene therapy clinical trial results: Potential impact of CpG sequences on gene expression. Blood, 2021; 137(6):763–774.

101.

Cao

, Dobrzynski

, Wang

, et al. Induction and role of regulatory CD4⁺CD25⁺ T cells in tolerance to the transgene product following hepatic in vivo gene transfer. Blood, 2007; 110(4):1132–1140.

102.

Coa

, Loduca

, and Herzog

. Role of regulatory T cells in tolerance to coagulation factors. J Thromb Haemostasis, 2009; 7(Suppl.1):88–91.

103.

Markusic

, Hoffman

, Perrin

, et al. Effective gene therapy for haemophilic mice with pathogenic factor IX antibodies. EMBO Mol Med, 2013; 5:1698–1709.

104.

Mátrai

, Cantore

, Bartholomae

, et al. Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigen-specific tolerance in mice with low genotoxic risk. Hepatology, 2011; 53(5):1696–1707.

105.

Hoffman

, Martino

, Sack

, et al. Nonredundant roles of IL-10 and TGF-β in suppression of immune responses to hepatic AAV-factor IX gene transfer. Mol Ther, 2011; 19:1263–1272.

106.

Burghardt

, Claass

, Erhardt

, et al. Hepatocytes induce Foxp3⁺ regulatory T cells by Notch signaling. J Leukoc Biol, 2014; 96:571–577.

107.

Carambia

, Freund

, Schwinge

, et al. TFG-β-dependent induction of CD4⁺CD25⁺Foxp3⁺ Tregs by liver sinusoidal endothelial cells. J Hepatol, 2014; 61:594–599.

108.

Faust

, Bell

, Zhu

, et al. The role of apoptosis in immune hyporesponsiveness following AAV8 liver gene transfer. Mol Ther, 2013; 21:2227–2235.

109.

Kumar

SRP

, Hoffman

, Terhorst

, et al. The balance between CD8⁺ T cell-mediated clearance of AAV-encoded antigen in the liver and tolerance is dependent of the vector dose. Mol Ther, 2017; 25(4):880–891.

110.

Chen

, Cai

, Li

, et al. A Treg-selective IL-2 mutein prevents the formation of factor VIII inhibitors in hemophilia mice treated with factor VIII gene therapy. Front Immunol, 2020; 11:638.

111.

Xiang

, Kuranda

, Quinn

, et al. The effect of rapamycin and ibrutinib on antibody responses to adeno-associated virus vector-mediated gene transfer. Hum Gene Ther, 2022; 33(11):614–624.

112.

Biswas

, Palaschak

, Kumar

SRP

, et al. B cell depletion eliminates FVIII memory B cells and enhances AAV8-coF8 immune tolerance induction when combined with rapamycin. Front Immunol, 2020; 11:1293.

113.

Nair

, De Wolf

, Nguyen

, et al. Gene therapy for hemophilia B using CB 2679d-GT: A novel factor IX variant with higher potency than factor IX padua. Blood, 2021; 137(21):2902–2906.

114.

Butterfield

JSS

, Yamada

, Bertolini

, et al. IL-15 blockade and rapamycin rescue multifactorial loss of factor VIII from AAV-transduced hepatocytes in hemophilia A mice. Mol Ther, 2022; 30(12):3552–3569.

115.

Fong

, Yates

, Sihn

, et al. Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A. Nat Med, 2022; 28(4):789–797.

116.

Handyside

, Ismail

, Zhang

, et al. Vector genome loss and epigenetic modifications mediate decline in transgene expression of AAV5 vectors produced in mammalian and insect cells. Mol Ther, 2022; 30(12):3570–3586.

117.

VandenDriessche

, Chuak

. Hemophilia “A” gene therapy: Lost in translation. Mol Ther, 2022; 30(12):3508–3509.

118.

Tran

, Lecomte

, Saleun

, et al. Human and insect cell-produced recombinant adeno-associated viruses show differences in genome heterogeneity. Hum Gene Ther, 2022; 33:371–388.

119.

Leibowitz

, Papathanasiou

, Doerfler

, et al. Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing. Nat Genet, 2021; 53(6):895–905.

120.

Ferrari

, Jacob

, Cesana

, et al. Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells. Cell Stem Cell, 2022; 29(10):1428–1444.

121.

Wagner

, Peter

, Schmueck-Henneresse

. Cas9-directed in humans—A model to evaluate regulatory T cells in gene therapy?. Gene Ther, 2021; 28(9):549–559.