AAV-mediated gene delivery
Encouraging News on Gene Therapy for Hemophilia A
Posted on by Dr. Francis Collins

About 20,000 people in the U.S. live with hemophilia A. It’s a rare X-linked genetic disorder that affects predominantly males and causes their blood to clot poorly when healing wounds. For some, routine daily activities can turn into painful medical emergencies to stop internal bleeding, all because of changes in a single gene that disables an essential clotting protein.
Now, results of an early-stage clinical trial, published recently in the New England Journal of Medicine, demonstrate that gene therapy is within reach to produce the essential clotting factor in people with hemophilia A. The results show that, in most of the 18 adult participants, a refined gene therapy strategy produced lasting expression of factor VIII (FVIII), the missing clotting factor in hemophilia A [1]. In fact, gene therapy helped most participants reduce—or, in some cases, completely eliminate—bleeding events.
Currently, the most-common treatment option for males with hemophilia A is intravenous infusion of FVIII concentrate. Though infused FVIII becomes immediately available in the bloodstream, these treatments aren’t a cure and must be repeated, often weekly or every other day, to prevent or control bleeding.
Gene therapy, however, represents a possible cure for hemophilia A. Earlier clinical trials reported some success using benign adeno-associated viruses (AAVs) as the vector to deliver the therapeutic FVIII gene to cells in the liver, where the clotting protein is made. But after a year, those trial participants had a marked decline in FVIII expression. Follow-up studies then found that the decline continued over time, thought to be at least in part because of an immune response to the AAV vector.
In the new study, an NIH-funded team led by Lindsey George and Katherine High of the Children’s Hospital of Philadelphia and the University of Pennsylvania, tested their refined delivery system. High is also currently with Asklepios BioPharmaceutical, Inc., Chapel Hill, NC. (Back in the 1970s, she and I were medical students in the same class at the University of North Carolina.) The study was also supported by Spark Therapeutics, Philadelphia.
Trial participants received a single infusion of the novel recombinant AAV-based gene therapy called SPK-8011. It is specifically designed to produce FVIII expression in the liver. In this phase 1/2 clinical trial, which evaluates the safety and initial efficacy of a treatment, participants received one of four different doses of SPK-8011. Most also received steroids to prevent or treat the presumed counterproductive immune response to the therapy.
The researchers followed participants for a year after the experimental treatment, and all enrolled in a follow-up trial for continued observation. During this time, researchers detected no major safety concerns, though several patients had increases in blood levels of a liver enzyme.
The great news is all participants produced the missing FVIII after gene therapy. Twelve of the 16 participants were followed for more than two years and had no apparent decrease in clotting factor activity. This is especially noteworthy because it offers the first demonstration of multiyear stable and durable FVIII expression in individuals with hemophilia A following gene transfer.
Even more encouraging, the men in the trial had more than a 92 percent reduction in bleeding episodes on average. Before treatment, most of the men had 8.5 bleeding episodes per year. After treatment, those events dropped to an average of less than one per year. However, two study participants lost FVIII expression within a year of treatment, presumably due to an immune response to the therapeutic AAV. This finding shows that, while steroids help, they don’t always prevent loss of a therapeutic gene’s expression.
Overall, the findings suggest that AAV-based gene therapy can lead to the durable production of FVIII over several years and significantly reduce bleeding events. The researchers are now exploring possibly more effective ways to control the immune response to AAV in expansion of this phase 1/2 investigation before pursuing a larger phase 3 trial. They’re continuing to monitor participants closely to establish safety and efficacy in the months and years to come.
On a related note, the recently announced Bespoke Gene Therapy Consortium (BGTC), a partnership between NIH and industry, will expand the refined gene therapy approach demonstrated here to more rare and ultrarare diseases. That should make these latest findings extremely encouraging news for the millions of people born with other rare genetic conditions caused by known alterations to a single gene.
Reference:
[1] Multiyear Factor VIII expression after AAV Gene transfer for Hemophilia A. George LA, Monahan PE, Eyster ME, Sullivan SK, Ragni MV, Croteau SE, Rasko JEJ, Recht M, Samelson-Jones BJ, MacDougall A, Jaworski K, Noble R, Curran M, Kuranda K, Mingozzi F, Chang T, Reape KZ, Anguela XM, High KA. N Engl J Med. 2021 Nov 18;385(21):1961-1973.
Links:
Hemophilia A (National Center for Advancing Translational Sciences/NIH)
FAQ About Rare Diseases (National Center for Advancing Translational Sciences/NIH)
Bespoke Gene Therapy Consortium (BGTC) (NIH)
Accelerating Medicines Partnership® (AMP®) (NIH)
Lindsey George (University of Pennsylvania, Philadelphia)
Katherine High (University of Pennsylvania)
NIH Support: National Heart, Lung, and Blood Institute
Engineering a Better Way to Deliver Therapeutic Genes to Muscles
Posted on by Dr. Francis Collins

Amid all the progress toward ending the COVID-19 pandemic, it’s worth remembering that researchers here and around the world continue to make important advances in tackling many other serious health conditions. As an inspiring NIH-supported example, I’d like to share an advance on the use of gene therapy for treating genetic diseases that progressively degenerate muscle, such as Duchenne muscular dystrophy (DMD).
As published recently in the journal Cell, researchers have developed a promising approach to deliver therapeutic genes and gene editing tools to muscle more efficiently, thus requiring lower doses [1]. In animal studies, the new approach has targeted muscle far more effectively than existing strategies. It offers an exciting way forward to reduce unwanted side effects from off-target delivery, which has hampered the development of gene therapy for many conditions.
In boys born with DMD (it’s an X-linked disease and therefore affects males), skeletal and heart muscles progressively weaken due to mutations in a gene encoding a critical muscle protein called dystrophin. By age 10, most boys require a wheelchair. Sadly, their life expectancy remains less than 30 years.
The hope is gene therapies will one day treat or even cure DMD and allow people with the disease to live longer, high-quality lives. Unfortunately, the benign adeno-associated viruses (AAVs) traditionally used to deliver the healthy intact dystrophin gene into cells mostly end up in the liver—not in muscles. It’s also the case for gene therapy of many other muscle-wasting genetic diseases.
The heavy dose of viral vector to the liver is not without concern. Recently and tragically, there have been deaths in a high-dose AAV gene therapy trial for X-linked myotubular myopathy (XLMTM), a different disorder of skeletal muscle in which there may already be underlying liver disease, potentially increasing susceptibility to toxicity.
To correct this concerning routing error, researchers led by Mohammadsharif Tabebordbar in the lab of Pardis Sabeti, Broad Institute of MIT and Harvard and Harvard University, Cambridge, MA, have now assembled an optimized collection of AAVs. They have been refined to be about 10 times better at reaching muscle fibers than those now used in laboratory studies and clinical trials. In fact, researchers call them myotube AAVs, or MyoAAVs.
MyoAAVs can deliver therapeutic genes to muscle at much lower doses—up to 250 times lower than what’s needed with traditional AAVs. While this approach hasn’t yet been tried in people, animal studies show that MyoAAVs also largely avoid the liver, raising the prospect for more effective gene therapies without the risk of liver damage and other serious side effects.
In the Cell paper, the researchers demonstrate how they generated MyoAAVs, starting out with the commonly used AAV9. Their goal was to modify the outer protein shell, or capsid, to create an AAV that would be much better at specifically targeting muscle. To do so, they turned to their capsid engineering platform known as, appropriately enough, DELIVER. It’s short for Directed Evolution of AAV capsids Leveraging In Vivo Expression of transgene RNA.
Here’s how DELIVER works. The researchers generate millions of different AAV capsids by adding random strings of amino acids to the portion of the AAV9 capsid that binds to cells. They inject those modified AAVs into mice and then sequence the RNA from cells in muscle tissue throughout the body. The researchers want to identify AAVs that not only enter muscle cells but that also successfully deliver therapeutic genes into the nucleus to compensate for the damaged version of the gene.
This search delivered not just one AAV—it produced several related ones, all bearing a unique surface structure that enabled them specifically to target muscle cells. Then, in collaboration with Amy Wagers, Harvard University, Cambridge, MA, the team tested their MyoAAV toolset in animal studies.
The first cargo, however, wasn’t a gene. It was the gene-editing system CRISPR-Cas9. The team found the MyoAAVs correctly delivered the gene-editing system to muscle cells and also repaired dysfunctional copies of the dystrophin gene better than the CRISPR cargo carried by conventional AAVs. Importantly, the muscles of MyoAAV-treated animals also showed greater strength and function.
Next, the researchers teamed up with Alan Beggs, Boston Children’s Hospital, and found that MyoAAV was effective in treating mouse models of XLMTM. This is the very condition mentioned above, in which very high dose gene therapy with a current AAV vector has led to tragic outcomes. XLMTM mice normally die in 10 weeks. But, after receiving MyoAAV carrying a corrective gene, all six mice had a normal lifespan. By comparison, mice treated in the same way with traditional AAV lived only up to 21 weeks of age. What’s more, the researchers used MyoAAV at a dose 100 times lower than that currently used in clinical trials.
While further study is needed before this approach can be tested in people, MyoAAV was also used to successfully introduce therapeutic genes into human cells in the lab. This suggests that the early success in animals might hold up in people. The approach also has promise for developing AAVs with potential for targeting other organs, thereby possibly providing treatment for a wide range of genetic conditions.
The new findings are the result of a decade of work from Tabebordbar, the study’s first author. His tireless work is also personal. His father has a rare genetic muscle disease that has put him in a wheelchair. With this latest advance, the hope is that the next generation of promising gene therapies might soon make its way to the clinic to help Tabebordbar’s father and so many other people.
Reference:
[1] Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species. Tabebordbar M, Lagerborg KA, Stanton A, King EM, Ye S, Tellez L, Krunnfusz A, Tavakoli S, Widrick JJ, Messemer KA, Troiano EC, Moghadaszadeh B, Peacker BL, Leacock KA, Horwitz N, Beggs AH, Wagers AJ, Sabeti PC. Cell. 2021 Sep 4:S0092-8674(21)01002-3.
Links:
Muscular Dystrophy Information Page (National Institute of Neurological Disorders and Stroke/NIH)
X-linked myotubular myopathy (Genetic and Rare Diseases Information Center/National Center for Advancing Translational Sciences/NIH)
Somatic Cell Genome Editing (Common Fund/NIH)
Mohammadsharif Tabebordbar (Broad Institute of MIT and Harvard and Harvard University, Cambridge, MA)
Sabeti Lab (Broad Institute of MIT and Harvard and Harvard University)
NIH Support: Eunice Kennedy Shriver National Institute of Child Health and Human Development; Common Fund
Snapshots of Life: Lighting up the Promise of Retinal Gene Therapy
Posted on by Dr. Francis Collins

Caption: Large-scale mosaic confocal micrograph showing expression of a marker gene (yellow) transferred by gene therapy techniques into the ganglion cells (blue) of a mouse retina.
Credit: Keunyoung Kim, Wonkyu Ju, and Mark Ellisman, National Center for Microscopy and Imaging Research, University of California, San Diego
The retina, like this one from a mouse that is flattened out and captured in a beautiful image, is a thin tissue that lines the back of the eye. Although only about the size of a postage stamp, the retina contains more than 100 distinct cell types that are organized into multiple information-processing layers. These layers work together to absorb light and translate it into electrical signals that stream via the optic nerve to the brain.
In people with inherited disorders in which the retina degenerates, an altered gene somewhere within this nexus of cells progressively robs them of their sight. This has led to a number of human clinical trials—with some encouraging progress being reported for at least one condition, Leber congenital amaurosis—that are transferring a normal version of the affected gene into retinal cells in hopes of restoring lost vision.
To better understand and improve this potential therapeutic strategy, researchers are gauging the efficiency of gene transfer into the retina via an imaging technique called large-scale mosaic confocal microscopy, which computationally assembles many small, high-resolution images in a way similar to Google Earth. In the example you see above, NIH-supported researchers Wonkyu Ju, Mark Ellisman, and their colleagues at the University of California, San Diego, engineered adeno-associated virus serotype 2 (AAV2) to deliver a dummy gene tagged with a fluorescent marker (yellow) into the ganglion cells (blue) of a mouse retina. Two months after AAV-mediated gene delivery, yellow had overlaid most of the blue, indicating the dummy gene had been selectively transferred into retinal ganglion cells at a high rate of efficiency [1].