Gene therapy carries immense potential for treating a range of genetic and acquired diseases due to its ability to replace or inactivate disease-causing genes. Many gene therapies rely on small, nonpathogenic viruses known as adeno-associated viral vectors (AAVs) to deliver genes into cells. Currently, there are five FDA-approved AAV gene therapies: Luxturna for inherited retinal dystrophy, Zolgensma for spinal muscular atrophy type 1, Hemgenix for haemophilia B, Elevidys for Duchenne muscular dystrophy, and Roctavian for severe hemophilia A. Despite significant advances in gene therapy development, key hurdles remain, including enhancing the delivery of AAVs. In a Drug Discovery News seminar, scientists discussed innovative strategies for overcoming these and other challenges and presented some exciting new applications for AAV gene therapies.  

A professional headshot of a woman with light brown hair wearing glasses, a white lab coat, a pink blouse, and a red beaded necklace with a large pendant.

Dawn Bowles is an assistant professor in surgery at Duke University. Her research team develops gene therapies to improve cardiac transplant outcomes.

credit: Dawn E. Bowles

Fixing a broken heart 

Dawn Bowles, a microbiologist at Duke University and the first seminar presenter, discussed using AAV gene therapy to improve heart transplant outcomes. Her long-term goal is to use AAV gene therapy to express molecules that suppress the immune response to prevent organ rejection and reduce transplant failure. This would eliminate the need for immunosuppressive drugs after a heart transplant and protect the transplanted organ from immune attack. “That is definitely an unmet need because of the negative impact of chronic immunosuppression on the recipient,” said Bowles. 

In a proof-of-concept experiment, Bowles’ group used an ex vivo reperfusion device to inject a donor pig heart with AAVs that carried the luciferase gene. The device maintained the pig donor heart in a near physiological state and permitted multiple passes of the gene therapy vector to maximize delivery before transplant. After two hours of perfusing the heart, they achieved stable luciferase expression that lasted for days in the recipient animal with no off-target effects or luciferase expression in other organs. They plan on using ex vivo gene therapy to deliver viral vectors that improve donor heart function before transplantation. 

The team is also exploring ex vivo gene therapy for auto transplantation to reverse or prevent heart failure. This involves removing the patient’s heart, delivering the gene therapy within the device, and putting it back into the patient.

Tuning the immune response 

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Samira Kiani is an associate professor of pathology and bioengineering at the University of Pittsburgh, working on strategies for safe gene therapies.

credit: Samira Kiani

Beside preventing the body from attacking a transplanted organ, inhibiting the immune response would also improve gene therapy efficacy and make gene therapy available to more people. 90 percent of the human population has already been exposed to one or more AAV serotypes, so many patients are not eligible for gene therapy because they have pre-existing immunity to the viral vector. In other patients, the gene therapy triggers an immune response that may render the therapy ineffective. “40 to 50 percent of patients that can otherwise be included in gene therapy trials are excluded because of circulating antibodies that recognize these viruses and disable them,” said Samira Kiani, a bioengineer at the University of Pittsburgh and second seminar presenter. 

To tackle this problem, Kiani’s group used zinc fingers specifically engineered to suppress the Myeloid differentiation primary response 88 (MyD88) gene, a crucial element in immune response activation. This removes the immune response towards AAV capsid antigens, which trains the immune system to ignore or tolerate them. During her presentation, Kiani discussed her team’s preliminary data indicating that lipid nanoparticles carrying zinc fingers successfully suppressed MyD88in  vivo, decreasing the antibody response against AAVs. “The hope is that by instructing the immune system to ignore gene therapy viruses, we will have lifesaving therapies accessible to those patients who are excluded from clinical trials because of high pre-existing antibodies,” said Kiani.  

Designing AAVs

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David Schaffer is a Hubbard Howe professor at the University of California, Berkeley, who uses directed evolution to engineer targeted and efficient viral gene therapy vectors.

credit: David Schaffer

Scientists are also taking advantage of the fact that AAV capsids are amenable to genetic engineering for displaying favorable characteristics such as reduced immunogenicity or enhanced gene therapy delivery. During the third presentation, David Schaffer, a chemical engineer at the University of California, Berkeley, discussed directed evolution, a process through which scientists introduce random mutations to wild type AAV capsids and subject them to an iterative selection process to identify those with the desired traits. “We have over a billion new engineered versions of AAVs, any of which may be a solution to a gene therapy problem that we’re trying to solve,” said Schaffer. 

He discussed applying directed evolution for enhancing AAV gene therapy delivery in macular degeneration, a disease in which the high expression of vascular endothelial growth factor (VEGF) kills photoreceptor neurons and leads to blindness. Traditional treatments were safe and effective but involved multiple eye injections per year, which limited patient compliance. To circumvent this problem, Schaffer’s team engineered AAVs capable of penetrating the retina and reaching the photoreceptor neurons. The AAVs encoded aflibercept, a VEGF inhibitor. A Phase 1 study in early 2022 showed that a single dose of this gene therapy eliminated the need for anti-VEGF injections in five patients. AAV genetic diversification and selection allowed Schaffer’s research team to engineer vectors for eight human clinical trials that are currently underway. 

Improving vector safety 

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Weidong Xiao is a professor of pediatrics at Indiana University who investigates gene therapy vectors and develops treatments for hemophilia.

credit: Weidong Xiao

Successfully engineered AAV capsids that display desirable traits start with high quality wild type capsids. Many AAV vectors contain DNA defects that pose a safety risk. During the last talk of the seminar, Weidong Xiao, molecular virologist at Indiana University, discussed how his team deciphers the origins of diverse AAV genomes and how these origins affect vector functionality. He discussed a study from another research group in which scientists gave mice an empty vector with no gene. They found that the empty vector integrated into the mice’s genes, significantly increasing the chance of liver cancer in cancer prone mice. Xiao’s group discovered that replication errors or recombination events during AAV packaging unintentionally introduced unusual structures in the AAV vectors called “snapback” genomes (SBG). These unusual structures were similar to the empty vectors that caused cancer in the mice and might have resulted from the binding of fragments from both strands of the vector DNA genome. “Snapback genomes exist in every vector preparation, and their amount varies,” said Xiao. “This suggests the potential for the existence of bad particles within our vector preparations.” Changing the orientation of the genes in an AAV vector could potentially prevent binding and make the vectors safer. 

Bowles’, Kiani’s, Schaffer’s, and Xiao’s work exemplifies the strides that scientists are making to solve key gene therapy challenges, including preventing vector immunogenicity, enhancing delivery, and improving safety. As more gene therapies become part of the pool of available treatments, new challenges will emerge. 

“Now that we have five FDA approved AAV gene therapies, we’re going to need to manufacture a ton of vector,” said Schaffer. This is a significant lingering challenge. Bowles agreed, “The Achilles heel is manufacturing. I think that’s going to be limiting us in all of these applications that we’re developing.” 

To learn more about Bowles’, Kiani’s, Schaffer’s, and Xiao’s work on overcoming the challenges of gene therapy, view the on-demand seminar on the Drug Discovery News website.


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