Data Show Potential of Roctavian Gene Therapy for Hemophilia A

Gene constructs making up Roctavian, an investigational gene therapy for hemophilia A, did not permanently integrate into the genome of non-human primates or cause any signs of tumors or malignancy in the animals. 

These are key findings from preclinical studies that BioMarin Pharmaceutical, the therapy’s developer, presented at the 24th American Society of Gene and Cell Therapy (ASGCT) Annual Meeting, held online May 11–14.

“BioMarin is committed to furthering our scientific understanding of gene therapy through our experience developing [Roctavian],” Lon Cardon, PhD, senior vice president and chief scientific strategy officer at BioMarin, said in a press release.

“This foundation of knowledge will help to inform the development of other gene therapies in our pipeline,” he added. “The studies presented at ASGCT illustrate our continued quest to define who responds to treatment, why, and for how long.”

Roctavian (valoctocogene roxaparvovec) uses a harmless adeno-associated virus (AAV) vector, called AAV5, to deliver a functional copy of the F8 gene, which contains instructions for making a blood clotting protein, called factor VIII (FVIII). This protein is missing or is defective in people with hemophilia A, causing patients to experience excessive and prolonged bleeds that can happen spontaneously.

By providing patient cells with a functional copy of F8, Roctavian is thought to restore the production of FVIII, and lower the risk of spontaneous bleeds.

Previous studies have shown that treatment with Roctavian led to a significant variability in the expression, or activity, of the delivered F8 gene between individuals across trials and in different animal models.

In an oral presentation, titled “Investigating Mechanisms of Variability of AAV5-hFVIII-SQ Expression in Mice” (abstract No. 68), researchers explored some of the factors that may affect gene expression and protein production in mice.

Healthy male mice received a single dose of Roctavian (AAV5-hFVIII-SQ) given directly into the bloodstream, and their FVIII DNA, RNA, and protein levels, along with other markers, were assessed. RNA is the intermediate molecule generated from DNA that serves as a template for protein production. 

A significant correlation was found between the levels of liver AAV5 vector DNA and FVIII protein, suggesting vector uptake and processing in liver cells might “be an important contributing factor.”

When higher doses of the AAV5 vector were used, the investigators also found a strong correlation between the levels of liver Grp78, a protein responsible for folding and transporting other proteins out of cells, and FVIII protein levels in the blood.

This suggested that “individuals who have a greater intrinsic ability to fold FVIII protein may secrete higher levels of mature protein into circulation,” the researchers wrote.

Other factors identified that might contribute to gene expression variability included pre-dosing cholesterol levels, expression patterns of the AAV receptor, and DNA repair mechanisms. 

“Overall, we demonstrated that [Roctavian] intersubject variability may be driven by multiple contributing host-mediated mechanisms,” the team concluded. “Additional studies that further investigate the mechanistic drivers of AAV5 gene therapy variability are ongoing.”

DNA constructs carried by AAV viral vectors usually do not permanently integrate into a patient’s genome — the DNA sequence of all human genes. However, sporadic integrations may still occur at low levels. Thus, there is a need to understand these events and their impact on a therapy’s effectiveness and safety.

In a poster, titled “Rare Genomic Integrations of AAV5-hFVIII-SQ Occur without Evidence of Clonal Activation or Gene-Specific Targeting” (abstract No. 895), researchers focused on assessing the prevalence and nature of these events in non-human primates.

To that end, they collected liver samples and analyzed DNA from 12 male non-human primates, either 13 or 26 weeks following treatment with Roctavian, which was given at different dose levels. 

No signs of DNA integration were found for more than 99.9% of the vectors used. Rare integration events occurred on average in less than 1 in 600 liver cells, a frequency in line with “expectations for AAV vectors and several orders of magnitude lower than the annual rate of natural mutations in humans,” the researchers wrote.

They found no evidence of cells with integrated DNA growing and expanding abnormally, “suggesting that individual integration sites were restricted to single cells or small groups of progeny cells.” Moreover, integration sites were distributed across the whole genome, and no pre-cancerous lesions, tumors, or malignancies were noted.

“Continuous scientific evaluations are required to better characterize integration profiles across AAV gene therapy products, improve our understanding of potential impact, and elucidate product-specific risk factors,” the team said. 

AAV-based gene therapies may elicit immune responses against viral components, as well as the viral genome, or the delivered FVIII protein product. Preclinical studies have suggested that treatment with immune-suppressing corticosteroids may affect these immune responses, leading to a long-lasting impact on protein expression.

Earlier work also showed prednisolone treatment started one week after Roctavian administration did not alter FVIII expression in mice.

In another poster, titled “The Effect of Prophylactic Corticosteroid Treatment on Adeno-Associated Virus (AAV)-Mediated Gene Expression” (abstract No. 338), investigators set out to explore whether corticosteroid administration prior to gene therapy might prevent early immune responses and promote the expression of gene constructs in mice.

Animals received oral prednisolone daily (2 mg/kg) for a total of four weeks, starting either one day or two hours before treatment with an AAV5 vector carrying a human a1-antitrypsin gene, which encoded the protein hA1AT. Control mice received water instead of prednisolone. 

In both prednisolone pre-treatment groups, levels of hA1AT protein in the bloodstream were significantly higher — 1.5 to 2.2 times higher — than non-prednisolone groups through 12 weeks. Likewise, the number of liver cells containing vector DNA was also higher in prednisolone-treated mice compared to controls.

Higher levels of full-length vector DNA significantly correlated with elevated hA1AT protein levels in the pre-treated groups compared to the untreated groups. Finally, researchers also found a trend towards less variability between individual mice in the prednisolone-treated groups.  

“Overall, corticosteroid use prior to AAV treatment increased liver-directed AAV expression that is associated with higher levels of full-length vector genomes in mice and may represent a potential strategy for the reduction of interindividual variability in AAV-mediated gene therapy,” the team concluded. 

BioMarin is hoping to submit a marketing authorization application for Roctavian in the European Union in June, and a biologics license application in the U.S. as early as April 2022, after regulatory agencies requested more clinical data.

“Our presentations at ASGCT extend our nearly decade-long experience in AAV scientific and clinical research to improve our understanding of the critical features of vectors, promoters, pharmacologic agents, and manufacturing technologies to help us deliver potentially transformative treatments to people with rare genetic disease,” Cardon said.