This year’s WFH World Congress featured a session where scientists involved in gene therapy approaches to hemophilia provided updates on developments in the delivery of therapies via stem cells, genome editing and liver-directed AAV vectors. David Lillicrap from Queen’s University, Canada, was the chair of this session and summarized the momentum of clinical gene therapy as remarkable.
Christopher B. Doering, Emory University, U.S.A., took the audience through the process of using stem cells in gene therapy for hemophilia A. Stem cell technology was first applied to T cells in the 1990s, but safety concerns necessitated a return of the research back to academic laboratories, said Doering.
“Stem cells are rare populations of unspecialized cells that are self-renewing and can become other cells,” began Doering. “Donor cells from a non-affected individual (from the blood) are transplanted into the patient. In order to apply [this approach] to hemophilia, this may require gene transfer.” Doering added that it is sometimes possible to use the patient’s own cells, harvested peripherally or from bone marrow. In order to implant stem cells, some of the patient’s cells must first be destroyed in order to make “space” for the new cells.
Challenges to combining gene transfer and stem cell technologies in hemophilia include the optimization of transgene expression and product biosynthesis, safe and effective pretransplantation conditioning, and clinical vector manufacturing, said Doering. “Stem cells can last for the lifetime of an individual so we have a potential cure. We need only to target a few cells as each stem cell will produce hundreds of daughter cells.”
A pilot clinical trial design has been approved by the US Food and Drug Administration, starting with a single site trial at Emory University, U.S.A.
Matthew Porteus, Lucille Packard Children’s Hospital, U.S.A., explained that genome editing is a method to correct disease causing variants. “This is a precise, controlled mutagenesis of the genome. Creating a break in the DNA will cause the cells to look for this and make a repair. So we can stimulate mutations at the site of the break.”
The repair could change the DNA sequence to one that already exists in the genome or to something novel using synthetic biology. This second option “creates a new therapeutic phenotype in the cell. In hemophilia it might be used to overexpress a clotting factor,” said Porteus.
Homologous recombination to change single nucleotide variants involves delivering an adeno-associated virus (AAV) nanoparticle. “In research with sickle cell disease there is about a 20 percent success rate. We can also insert a gene cassette into a safe harbor or single location in the genome,” said Porteus.
“Targeting transgene addition without knocking out the target gene or knocking a gene into being highly expressed has some exciting applications,” noted Porteus. “There are opportunities and challenges for in vivo gene editing for hemophilia. There would be no need to give patients conditioning agents and it is a potential method to edit cells that naturally make clotting factors.” One drawback however, is a relative inability to monitor efficacy and off-target effects, he added.
Porteus noted that an ethical concern of genome editing is equity and distribution and how to make it available to people with hemophilia in all parts of the world.
Brigit E Riley, Sangamo BioSciences, U.S.A., delivered new data on FVIII gene therapy via AAV delivery. “Using AAV in clinical and preclinical trials for FIX has been successful, however there is a lag in the clinic for FVIII.”
She said liver-directed AAV FVIII cDNA gene therapy is being explored. Recombinant AAV is efficient and stable long-term in tissues that do not divide such as the liver, brain, and muscle. This provides the potential for long-term production of a gene of interest in the liver. FVIII, however, is not an ideal gene for delivery via AAV as it is constrained by its large gene size and a low efficiency of transcription/translation. These challenges can be partially overcome by multifactorial modifications. “With the modifications, virus yield was improved 8 to 10 fold,” said Riley.
She noted that data from in vitro experiments show good correlation between FVIII activity and levels over a range of doses. In vivo, expression of the hFVIII (humn FVIII) cassette from the AAV vehicle in wild-type mice resulted in FVIII expression at twice the normal physiological level. Further experiments in a hemophilia A mouse model demonstrated in vivo FVIII activity three times normal physiological levels with levels remaining stable over time. A reduced bleeding time was also observed. In nonhuman primates, similar in vivo experiments resulted in FVIII at four to sixtimes normal physiological levels. “Follow-up dose finding studies are aimed at determining minimal dose,” said Riley.
Despite all of these exciting advances, the challenge of financial incentives remains to be addressed. Porteus pondered, “With no established reimbursement model for a one-time gene therapy, it begs the question, ‘Are stakeholders willing to take a chance on experimental curative therapies that have a different conceptual basis, when the current paradigm has transformed the lives of hemophilia patients?’”