An increasing number of studies support the use of gene therapy in patients with hemophilia. For instance, evidence shows that liver-directed gene therapy has the potential to transform congenital hemophilia from an incurable disease with a severe phenotype into a moderate or even mild type of hemophilia.

However, several factors restrict the wide application of gene therapy in patients with hemophilia, as discussed by Rodríguez-Merchán in a review article published in the International Journal of Molecular Sciences. These include unknown risks and long-term outcomes, mainly attributed to the use of gene transfer vectors, and the lack of experience of healthcare providers in managing putative adverse events.

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Rodríguez-Merchán also highlight the clinical use of very effective palliative treatments that could improve patients’ quality of life when administered prophylactically. These include extended half-life recombinant products and new non-replacement-based therapies. In fact, in light of the current knowledge, it is difficult to anticipate the proportion of patients who would benefit from gene therapy.

Adverse Events and Hepatotoxicity

Gene therapy protocols aim to transfer a DNA fragment that encodes the protein of interest into cells. They are usually more effective than those of cell therapy. However, they are also associated with a greater number of adverse events.

Adverse events in gene therapy are mainly related to the vector, which can be of viral or nonviral origin. “An ideal transfer vector should be immunologically inert; highly tissue- and cell-specific; integrative; capable of sustainably producing the transgene and transducing genes to divided or undivided cells; easy to produce at large scale; and capable of maximum transgene loading,” Rodríguez-Merchán explained. However, and despite recent improvements, immunogenicity, hepatotoxicity, and insertional mutagenesis of transfer vectors are still major challenges.

Gene Therapy Reduces Bleed Rates Among Patients With Hemophilia A

The most commonly used viral vectors in gene therapy for hemophilia are of the recombinant type, such as adeno-associated virus (AAV) vectors, which display tropism for several target tissues (eg, liver). These are followed by in vivo and ex vivo lentiviral vectors, which are integrative and less prone to lead to insertional mutagenesis, significant immune response, or hepatotoxicity.

Though AAVs usually present with a good safety profile, some serotypes are more immunogenic and exhibit hepatotoxicity more than others. Therefore, transient immunosuppressive techniques have been employed to blunt cells’ immune response against the virus.

Immunosuppression is of great importance in the context of AAV-mediated gene therapy since pre-existing antibodies against AAVs have a negative impact on the effectiveness of the therapeutics. However, immunosuppressive drugs increase the overall risk of infection and might result in several metabolic side effects. Therefore, efforts have been made to identify less immunogenic AAV serotypes.

A Mixed Bag of Clinical Results

The first human trials of AAV vector-based gene therapy showed it enabled the expression of clotting factors in the liver at therapeutic levels. This expression was, nonetheless, short-lived because of the generation of a cytotoxic immune response against the vector-transduced hepatocytes.

These initial studies also disclosed the need for screening hemophilia patients for pre-existing anti-AAV neutralizing antibodies and uncovered the limitations of previous animal studies, which failed to predict such problems.

Later studies showed that the combination of hepatotropic AAV serotypes and oral corticosteroid-based transient immunomodulation led to sustained (at least 10 years) expression of human coagulation factor IX in hemophilia patients. These studies still detected an immune response directed against the AAV capsid in patients receiving the highest treatment dose.

Investigational Hemophilia Treatment Receives Orphan Drug Designation

In patients with hemophilia B, not all studies assessing gene therapy have been successful in achieving long-term expression. For instance, BAX 335, an AAV8 vector expressing FIX Padua (a naturally occurring missense variant with increased activity) was evaluated in a phase 1/2, open-label, dose-escalation study (NCT01687608).

“Measurable FIX Padua transgene expression was documented in seven of the eight participants, although only one participant achieved sustained therapeutic FIX at approximately 20% of normal, with no bleeding episodes, in the absence of replacement therapy over the 4 years of follow-up,” Monahan et al explained in a review article published in the Journal of Clinical Medicine. In the other participants, “the initial circulating FIX activity was not sustained beyond 5-11 weeks.”

More recently, techniques for vector codon optimization had allowed reducing the potential for triggering innate immune responses by decreasing the content of CpG motifs. This seems to help manage vector-associated liver inflammation. For instance, relatively low doses of an AAV vector expressing the FIX Padua transgene achieved an average FIX activity of 33.7 ± 18.5% in a phase 1/2, open-label, nonrandomized, dose-escalation study (NCT02484092). This led to reduced bleeding in 7 participants who were previously on prophylaxis. Out of 10 participants, 2 experienced an immune-mediated increase in liver enzymes, which was managed with the administration of oral corticosteroids.

Another phase 1/2 study (NCT02396342) evaluated the use of a wild-type FIX expression construct packaged into an AAV5 vector. In this study, 20%-40% of participants developed limited, asymptomatic, and transient increases in alanine aminotransferase (ALT). These episodes were successfully treated with oral corticosteroids. During this period of liver transaminitis, the authors did not observe the loss of FIX expression or evidence of circulating T-cells targeting AAV capsid epitopes.

Hemophilia May Benefit From Redosable Gene Therapy

Clinical data is more scarce when it comes to hemophilia A. Even though, results are promising. A 3-year sustained transgene expression was observed in participants receiving an AAV vector that contained a codon-optimized, B domain-deleted factor VIII (FVIII) complementary deoxyribonucleic acid (cDNA). The study did not report any cases of inhibitor development, thromboses, or deaths. However, the authors found alterations in liver function tests that lasted several weeks.

“Additional hemophilia A gene therapy trial results have been reported in preliminary form only but when considered in aggregate raise several potential immunologic obstacles and unknowns that will guide future research and development,” Monahan et al wrote. FVIII expression was not associated with VIII inhibitor development in either of them. However, the researchers pointed to greater interindividual variability than in studies of gene therapy for hemophilia B.


Rodríguez-Merchán EC, De Pablo-Moreno JA, Liras A. Gene therapy in hemophilia: recent advances. Int J Mol Sci. 2021;22(14). doi:10.3390/ijms22147647

Monahan PE, Négrier C, Tarantino M, Valentino LA, Mingozzi F. Emerging immunogenicity and genotoxicity considerations of adeno-associated virus vector gene therapy for hemophilia. J Clin Med. 2021;10(11). doi:10.3390/jcm10112471