Gene Therapy and Bleeding Disorders: Where Are We Now, and What’s on the Horizon?

The National Hemophilia Foundation’s 16th Workshop on Novel Technologies and Gene Transfer for Hemophilia, held in November, addressed these questions and more.

Since 1966, leading immunologists, clinicians and researchers in gene therapy have been convening every one to three years to discuss the latest findings and obstacles in the search for possible genetic cures for bleeding disorders. Each time they have come together, they have concluded that while much has been learned, much more needs to be done.

The Science Community Does Not Stop for a Pandemic

In November, co-chairs Glenn Pierce, MD, PhD, and David Lillicrap, MD, along with the National Hemophilia Foundation’s (NHF) Medical Programs and Information Department, convened the 16th Workshop on Novel Technologies and Gene Transfer for Hemophilia in Washington, DC. The hybrid community of online and masked, socially distanced attendees from around the globe presented their latest bleeding disorder gene therapy research and challenged one another through thoughtful discussions.

The Origins of Gene Therapy

Once researchers discovered that DNA was the source of genetic inheritance and diseases, they began exploring the possibility of replacing a damaged gene. Today, more than 40 years of advancements have turned the hypothetical into probable for several medical conditions. As genetic sequencing became more affordable and available, scientists identified genetic markers for numerous conditions. However, the technology needed to safely deliver nucleic acid cargo inside cells has lagged behind the technology development used to identify the disease-associated genes.

Naturally occurring viruses (such as cold viruses) can deliver genetic material into cells, “tricking” them into making more copies. Scientists have taken advantage of this and devised adaptations to deliver gene therapy using “vectors” based on viruses. However, some viral vectors have produced serious adverse events, including cancer.

Gene therapy for hemophilia involves replacing the mutated F8 or F9 gene with a functioning copy of the gene so that the instructions for making clotting factor are not broken. Several techniques have been explored to determine which ones show the most promise with the fewest side effects, and scientists are completing research to evaluate the safety and efficacy of the potential treatments. Researchers have also begun exploring gene therapy for von Willebrand disease (VWD). While most gene therapy researchers hope to completely cure bleeding disorders, many recognize that even improving the body’s ability to make clotting factor may lessen disease severity and give people a better quality of life.

The most important aspects of gene therapy are efficacy and safety, followed by effectiveness and cost. The distinction is between how the treatment works in a controlled, ideal condition such as the research studies discussed at the workshop (efficacy) versus possible side effects that might occur once the therapy is used in the real world (effectiveness). These fundamentals are paramount to understanding what researchers have learned and where gene therapy is headed.

Fundamentals of Bleeding Disorders

Blood contains proteins (clotting factor) that help stop bleeding after an injury or surgery. Hemophilia, a rare blood disorder, involves low amounts of either factor VIII or factor IX clotting factor, which causes a person’s blood to not clot correctly—leading to nosebleeds, blood in the urine or stool, bleeding issues after an injury or surgery, or unexplained bleeding, pain, swelling or tightness in the joints. Extremely low levels of clotting factor indicate a more severe diagnosis and possible serious bleeding-related health problems.

The current preferred treatment for hemophilia is to replace the missing factor or to administer medicines that circumvent the need for the missing factor. People can learn to administer the treatment (clotting factor concentrates) by injecting them into a vein (infusing) to treat periodic bleeding episodes. Their healthcare provider may also prescribe regular prophylactic infusions to try to prevent bleeding episodes.

Workshop Highlights

The studies presented at the workshop that involved bringing a novel therapy from the lab to trials to the clinic were complex. The research is occurring in mice, primates and dogs. The presenters discussed gene therapy targeted at liver cells (hepatocytes) and bone marrow (intraosseous).

Although multiple vehicles (vectors) have been evaluated for targeting the liver for gene therapy in people with hemophilia, most of the discussion was about adeno-associated viruses (AAV) and lentiviral vectors.

1. Advancements in AAV Research

AAVs are small viruses that infect some primates, including humans. AAVs produce a mild immune response rather than cause disease, and different AAVs have affinity for different tissues. As of 2019, AAVs had been used in more than 250 gene therapy clinical trials across a variety of diseases, including hemophilia.

In laboratory and animal models, researchers identified the main mechanism leading to the formation of genome particles generated by the host cell environment. The scientists concluded that their results provide new clues for how to improve both vector efficiency and safety.

Another researcher shared that the differences between rodents and humans have become apparent in AAV clinical trials. Some findings in mouse models were replicated in humans, but others have poorly translated. Researchers still need to better understand how human liver cells (hepatocytes) can replace the mouse functional tissue of an organ (parenchyma). They also discussed limitations and strategies to improve preclinical AAV gene therapy mouse models. In addition, the researchers discussed their ongoing efforts to improve the ability of AAV vectors to target and infect specific cells.

Also, studies looking at AAV-transduced liver tissue under a microscope found that after a single infusion of gene therapy (AAV5-hFVIII-SQ), clinically significant factor VIII levels and reduced annualized bleeding occurred. Analyzing the therapy’s effect on the livers demonstrated no structural changes and mild fat buildup in the livers of four of five participants.

2. Advancements in Lentiviral Vectors

Researchers also explained that a human immunodeficiency virus 1 (HIV-1)-derived vehicle is the most used lentiviral vector in gene therapy. However, the production of lentiviral vectors has been challenging and low-yielding, and thus not cost-effective. One trial using lentiviral gene therapy with a corticosteroid immune-suppression regimen in nonhuman primates demonstrated efficient and well-tolerated liver gene transfer, with an improved therapeutic index for factor VIII.

Another study delivered lentiviral vectors into bone marrow, targeting factor VIII. Gene therapy delivery to the shin or hip bone of four dogs with hemophilia A resulted in factor VIII in the platelets of the treated dogs but no factor VIII expression in their plasma. All the treated dogs had reduced annual bleeding in the two years after gene therapy.

Researchers also evaluated gene transfer therapy into humanized mice’s stem cells that give rise to other blood cells. Injecting the lentiviral vectors into the bone marrow of humanized mice led to factor VIII platelet expression. The results indicate safety and efficacy in canine and humanized mice, adding evidence for translating the model to clinical trials.

The Genetics of Hemophilia

Because hemophilia is an inherited disorder, understanding genetics is important to understanding the potential impact of gene therapy. Genes provide instructions for making proteins. A mutation in the gene that gives the directions for clotting factor is typically passed from one generation to the next and may result in the blood not clotting properly. You inherit one chromosome from each parent. Females have XX chromosomes, and males have XY. The gene for factors VIII and IX are both on the X chromosome, so males will have symptomatic disease if their lone X chromosome has the hemophilia mutation. A father with the mutation passes it to his daughters when he gives his lone X chromosome to them. He would not pass the mutation to any sons, as the Y chromosome he passes does not have the mutation.

A female may have one X chromosome with the mutation and one without, so her unaffected X chromosome may signal enough clotting factor that she might not realize she is a carrier. A mother who has one affected X chromosome has a 50% chance of passing it to her offspring. It is rare for a mother to have two affected X chromosomes, but if she does, she will pass an affected X chromosome to 100% of her offspring.

3. Gene Therapy Research for VWD

While gene therapy research for hemophilia is in advanced stages, other bleeding disorders have not received the same attention. Von Willebrand disease research, in particular, has faced hurdles.

The complementary DNA (cDNA) that encodes von Willebrand factor (VWF) exceeds the compatible physical size for AAV delivery. Also, VWF is best expressed in the thin layer of cells that line the interior surface of all blood vessels (endothelial cells) rather than in liver cells (hepatocytes). Researchers are working on mechanisms to overcome these issues. Their use of a specific vector (dual-hybrid AAV9) to overcome size limitations resulted in long-term expression of VWF in lab studies of human endothelial cells and in VWF-deficient mice. However, they noted that the current VWF levels are too low to be clinically relevant and that additional engineering is needed to improve expression levels of VWF.

Pulling It All Together

Many lessons were learned during the workshop. However, many unknowns remain, including safety concerns regarding the liver, whose job is to regulate most chemical levels in the blood by producing proteins for blood plasma and cholesterol to help carry fats through the body. The liver also produces bile, which helps carry away waste and break down fats in the small intestine during digestion. Therefore, the researchers’ focus on maintaining liver health is to ensure the liver does not become severely damaged and can properly continue breaking down harmful substances and excreting the byproducts for removal from the body.

Several researchers at the workshop agreed that, rather than being a cure, gene therapy will probably reduce the severity of disease, requiring less factor therapy and improving quality of life. However, some people may have an immune response to the virus used for gene therapy, which would diminish the therapy’s effectiveness. The researchers also discussed how concerns about an unwanted immune response to the vector led to the incorporation of immunosuppression therapy into some clinical studies. In addition, the dose required to elicit the desired response is variable. The researchers agreed that work remains to safely translate the animal research to human applications.

For many workshop attendees, the most impactful presentation was a panel made up of two physicians, an NHF staff member and a graduate student, all of whom are familiar with healthcare and either live with or are a caregiver for a person with a bleeding disorder. The four panelists shared hopes and concerns for future treatments. Their questions reminded the room that some people may eventually be able to improve their quality of life significantly and either stop using or reduce their reliance on clotting factor products with the use of gene therapy—but that many people will not qualify for these treatments because of health concerns, the way their bodies respond, or simply a lack of access and the treatment’s high cost.

This somber note included a plea for continued research into treatment pathways beyond gene therapy to help those with bleeding disorders live their best lives possible.

Source: National Hemophilia Foundation, August 2022

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