Targeting Issues in AAV Vector Gene Therapy for Hemophilia

Hemophilia is a bleeding disorder affecting more than 1,125,000 people worldwide: three times higher than the previous estimate of 400,000.1 The disorder is caused by mutations of factor genes in hepatocyte cells of the liver, resulting in partial or total deficiencies of blood-clotting proteins. Physical symptoms range from severe bleeding after a major injury to spontaneous bleeding in muscles and blood build-up in joints, all of which limit the lifestyle and shorten the lifespan of those affected. Hemophilia currently has no cure. Treatments such as injecting clotting factor concentrates exist; however, such therapies require thousands of injections over a lifetime and cost an average of more than $270,000 annually. These factors only make it harder for hemophilia patients living in less industrialized nations or more rural areas to access adequate treatment. Researchers around the world are currently exploring alternative treatment methods, namely gene therapies using non-viral naked DNA or viral vectors. Such experimental techniques use protective molecules or human-modified viruses to transport and introduce genes into target cells. The end result is the creation of necessary proteins that correct mutations and alleviate disease symptoms. Of the two methods, viral vectors are more popular for liver gene therapy. At the University of North Carolina Gene Therapy Center, Dr. Chengwen Li and his team focus on the adeno-associated virus (AAV) for application in viral vector gene therapy. AAV has shown promising results in clinical trials for hemophilia, with patients’ blood-clotting factors increasing to therapeutic levels for long periods of time. Furthermore, in theory, AAV gene therapy has the potential to cure hemophilia with one injection, since the correct genetic code would stay in the human body for autonomous protein creation. This improves upon current treatment options, which involve multiple expensive injections that only alleviate hemophilia symptoms. Many of the AAV’s characteristics render it an attractive gene therapy vector. For example, the virus allows for long-term protein production in the cell, has the ability to infect a broad spectrum of cell types, invokes a relatively low but still present immune response, and lacks the ability to cause illness. 2 Despite its beneficial features, researchers like Dr. Li must continue to search for ways to modify the AAV genome in order to make it more suitable for effective disease treatment. Dr. Li and his lab mainly investigate how to overcome the immune response against AAV vectors, developing new strategies to enhance AAV vector transduction and specifying AAV vector gene therapy for rare diseases like hemophilia. Their most recent research addresses all three necessary improve-

By Kelly Fan

Figure 1. AAV vector transduction pathway, starting with the vector entering the cell and ending with the therapeutic protein, like clotting factor protein for hemophilia. ments to AAV gene therapy, with a focus on neutralizing antibodies called Nabs. The fact that “over 90% of humans have already gotten an AAV infection” presents a significant problem for hemophilia-centered AAV gene therapy. 3 Around 50% of previously-infected people generate Nabs, which recognize and block AAV capsids (the outer coating surrounding the genome) from interacting with host cells. As a result, transduction cannot occur and AAV gene therapy would not show many, if any, beneficial effects for hemophilia patients in that 50%. However, many varieties, or serotypes, of AAV exist. The various serotypes have different genetic material but similar protein structures with nuances impacting their interactions.

Certain AAV vector serotypes compared Nab activity to others might better escape the Nabs through the produced by an individual patient. The methods of basic theory provides the foundation directed evofor the directed evolution method. lution and usThus, Dr. Li and his team modified the ing chimeric AAV genome to change the capsid’s mice. With protein structures that interact with such positive Nabs and cells—DNase enzymes cut results, more DNA from different AAV serotypes into hemophilia small pieces, which are recombined to patients who form mutant AAVs. have Nabs At first, the Li Lab randomized the could be reprocess in order to create a library of dif- cruited for ferent mutants. Then, they injected mu- clinical trials tant AAV into chimeric mice with livers and treated Figure 3. Results of AAV neutralization for different AAV serotypes made up of human hepatocytes. 4 Dr. by AAV gene and mutant. AAV mutant LP2-10 required the highest concentration Li and his team used these humanized therapy, since of Nab in IVIG to have transduction abilities inhibited completely. mice to address the problem that “re- their immune searchers can take inspiration from such sults from animals cannot exactly repre- system would not react as strongly in an methods to develop more mutant AAV sent what will work in humans.” Certain attempt to eradicate AAV vectors. vectors able to travel to the brain, musserotypes may show great transduction The current major drawback to cles, and anywhere else in the body as in mouse cells in lower dosages, but ne- AAV LP2-10 is that its transduction ef- well as deliver therapeutic genes. cessitate much higher dosages to show ficiency in human hepatocytes is less A future where people from all the same level of transduction in hu- than the best serotypes used to create over the world with hemophilia can fimans. 3 Using hepatocyte-humanized the mutant AAV library, indicating a nally receive adequate treatments and mice may allow for smoother transition direction of future study for Dr. Li and live more full lives is possible with conof beneficial results from animal models his lab. The lab plans to use the mu- tinuous research and improvements in to human clinical trials. Once mutant tant AAV LP2-10 as a “template to build AAV gene therapy. Furthermore, those AAV vectors were extracted from the upon,” rationally targeting places in its possibilities are not only limited to hexenografted hepatocytes, the lab tested DNA genome to modify so that its he- mophilia. Dr. Li suggests hopes that as their transduction efficiency and resis- patocyte transduction is high enough research on AAV gene therapy improves tance to Nab activity. to have viable therapeutic effects on its effectiveness, “any disease in the fuDr. Li and other researchers iden- hemophilia. Alternatively, the lab could ture could possibly be treated by gene tified Mutant AAV LP2-10 as the mutant also do another round of direct evolu- therapy.” 3 capsid with the highest ability to escape Nabs and, accordingly, a better chance tion, creating a new mutant AAV library with the inclusion of AAV LP2-10 to “mix References 1.Iorio, A.; Stonebraker, J. S.; Chambost, H.; of transducing hepatocytes. Its capsid in its significant ability to escape Nabs.” 3 Makris, M.; Coffin, D.; Herr, C.; Germini, This may lead to the F.; Data and Demographics Committee creation of a better of the World Federation of Hemophilia. vector with a higher Establishing the Prevalence and Prevalence transduction effi- at Birth of Hemophilia in Males: A ciency for hepatoMeta-analytic Approach Using National Registries. Ann Intern Med 2019, 171(8), cytes. 540-546. https://www.acpjournals.org/doi/ The results full/10.7326/M19-1208 are not specific to 2.Li, C.; Samulski R.J. Engineering adenohemophilia. Re- associated virus vectors for gene therapy. searchers can also Nat Rev Genet 2020, 21, 255–272. https://doi. Figure 2. The left shows wild type AAV being inhibited by neutralizing antibodies once in the body. The right displays an apply Dr. Li’s findings to other liver diseases because org/10.1038/s41576-019-0205-4 3.Interview with Chengwen Li, Ph.D. 09/11/2020 4.Pei, X.; Shao, W.; Xing, A.; Askew, C.; overview of the directed evolution process. AAV LP2-10 widely Chen, X.; Cui, C.; Abajas, Y. L.; Gerber, surface structures can escape higher avoids Nabs in human bodily fluids. Ad- D. A.; Merricks, E. P.; Nichols, T. C.; et al. concentrations of Nabs than other se- ditionally, the methods of direct evo- Development of AAV Variants with Human rotypes and mutants.4 The outcomes lution, viral libraries, and humanized Hepatocyte Tropism and Neutralizing Antibody Escape Capacity. Mol Ther prove it is possible to isolate mutant mice have the potential to further gene Methods Clin Dev 2020, 18, 259–268. AAV vectors with the ability to evade therapy research on more diseases. Re- https://doi.org/10.1016/j.omtm.2020.06.003 45

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