Successful Gene Therapy Treatment and Redosing in an Animal Model of Methylmalonic Acidemia
Petr O. Ilyinskii and Takashi Kei Kishimoto
Selecta Biosciences, Watertown, MA
Gene therapy is making a difference in the lives of patients with previously untreatable genetic diseases. Most of these rare diseases result from a defect in a single gene. Gene therapy aims to deliver working copies of the gene into affected organs and tissues to correct the underlying genetic defect. The potential tools for such targeted gene delivery have been studied for many years in animal models, but now are finally coming to clinical fruition. This includes the possibility of treating inborn errors of metabolism, especially those involving the liver, such as methylmalonic acidemia (MMA). The majority of individuals with MMA have a deficiency in a gene called methylmalonic CoA mutase, or MMUT for short, which results in the body’s inability to process certain fragments of proteins and fats. This metabolic defect can lead to a build-up of toxic substances causing bouts of serious illness called metabolic crises. The liver is a key organ involved in this processing of proteins and fats and therefore delivery of a fully functional MMUT gene into the liver has the potential to greatly reduce the severity of disease and improve quality of life. This line of thinking is further supported by the clinical benefit that many MMA patients exhibit after successful liver transplantation. The advantage of gene therapy is that it does not require the availability of a suitable organ to transplant or complex surgery, nor does it require lifelong immune suppression.
The use of adeno-associated virus (AAV) vectors is one of the most advanced and extensively studied approaches to deliver working copies of genes. It has been studied in animal models of MMA and used extensively in human gene therapy clinical trials for other genetic diseases. AAV itself is a harmless virus. The modified AAV used for gene therapy cannot make copies of itself because the genes of the virus have been removed and replaced with a therapeutic gene, such as MMUT. Notably, the outer structure of the virus particle, called the capsid, allows the therapeutic AAV vector to enter the target cell and to unload its genetic cargo (i.e. the correct version of the MMUT gene). Some types of AAV selectively target liver cells, which enables targeted delivery of the MMUT gene to the liver cells. After unloading of the MMUT gene, the AAV capsid is degraded but the MMUT gene stays within the target cell. The new MMUT gene uses the liver cell’s machinery to produce the correct version of the MMUT enzyme, which restores the ability of the cell to break-down protein and fats and reduce the buildup of methylmalonic acid and other toxic metabolites. This strategy has been demonstrated in mouse models of MMA in the laboratory headed by Dr. Charles Venditti at the National Human Genome Research Institute (NHGRI).
Metabolic disorders, such as MMA, can affect a child from birth. Most cases of severe MMA require immediate management and early intervention to prevent metabolic crisis and minimize tissue and organ damage. Despite best efforts, patient management is very challenging due to the variety of stressors (e.g. diet, infection) that can trigger metabolic crisis. Thus, gene therapy is likely to provide the most benefit when applied at an early age.
One challenge with treating young children is that the AAV vector is non-replicating, and gene therapy delivers only a limited number of MMUT gene copies into the body. Over time, as the child grows, their liver cells will continue to multiply, but the number of transferred MMUT gene copies will not. Therefore, the therapeutic benefit may decrease over time if the remaining MMUT gene copies cannot keep up with the metabolic demands of a growing child. Thus, it is possible that an MMA patient treated with AAV-MMUT as a child will require additional therapeutic treatment during their lifetime. The ability to re-dose AAV could provide significant benefits to patients, including re-dosing to help more patients to achieve therapeutic benefit and redosing to restore activity in patients in patients that have lost therapeutic benefit (Figure 1).
Figure 1. Re-dosing has the potential to provide therapeutic benefit to those patients that fail to achieve therapeutic activity after the first dose (Top). Re-dosing could also help to restore therapeutic activity to those patients that have lost therapeutic benefit (Botom).
However, there are significant biological barriers to re-treatment with AAV gene therapy vectors due to the patient’s immune response to the AAV capsid. Although the AAV vector is no longer a virus, it is dressed in the coat of a virus (the capsid), and thus treated by the immune system as a potential threat. The immune system produces antibodies against the AAV capsid, much like it would produce antibodies to an actual virus or to a vaccine. These antibodies circulate in the blood and stand ready to block the AAV vector by binding to the capsid surface and tagging it for elimination by the immune system. These circulating antibodies will form after the first treatment with AAV and effectively prevent the ability to re-administer AAV gene therapy.
Selecta Biosciences has developed a technology called ImmTOR that is designed to selectively prevent unwanted immune responses to AAV vectors and other biologic drugs. ImmTOR consists of nanoparticles containing a drug called rapamycin (a well-known and FDA approved drug used to prevent organ rejection in patients receiving kidney transplants). In contrast to rapamycin, which requires life-long daily administration to prevent organ rejection, ImmTOR is designed to be dosed around the time of AAV administration to inhibit the immune response to the AAV capsid by selectively inducing immune tolerance. Immune tolerance is the process by which the immune system regulates itself to prevent unwanted immune responses to the body’s own proteins.
The immune system can ‘learn’ to become tolerant of other harmless proteins, such as dietary proteins or proteins from bacteria that normally colonize the gut. ImmTOR facilitates specific immune tolerance induction when co-administered with biologic drugs, such as AAV vectors. Animal studies have shown that ImmTOR inhibts immune responses to AAV vectors enabling successful re-administration of gene therapy. ImmTOR has also been studied in humans in combination with an enzyme therapy called pegadricase to treat severe gout, a non-genetic disease. Like AAV, pegadricase is a highly immunogenic drug. When used alone, pegadricase induces strong antibody response and cannot be re-dosed, but when combined with ImmTOR it has been successfully administered up to 6 times in humans. Over 250 patients have been treated with ImmTOR, which is currently in Phase 3 clinical trials, the last phase of clinical testing prior to marketing.
Based on this knowledge, scientists from Selecta Biosciences and NHGRI decided to test if treatment with AAV-MMUT, an AAV vector designed to treat MMA, combined with ImmTOR would suppress the development of antibodies against AAV and thus allow for vector re-dosing. Separately, Selecta scientists have recently shown that co-administration of liver-targeting AAV vectors and ImmTOR also led to an increase in the expression of the AAV-delivered gene after just a single treatment. However, this was not done using a therapeutic gene such as MMUT. Thus, there were two separate questions that scientists were trying to answer in the new study: 1) Would there be a beneficial effect of ImmTOR on initial AAV administration in a mouse model of human MMA disease? 2) Would co-administration of AAV-MMUT and ImmTOR decrease immune responses to AAV and, consequently, enable therapeutic redosing? It is important to mention that re-dosing of AAV gene therapy and ImmTOR in an animal model of human disease had never been tested before.
A combination of ImmTOR and AAV-MMUT was administered to MMA mice developed at NHGRI. The mouse model used allowed the study of AAV gene therapies in juvenile mice, as a model for gene therapy in young children4. Mice were treated at approximately 4 weeks of age (roughly correlating to a child 3-8 years of age) and then again 8 weeks later (when the mouse becomes a young adult) with AAV-MMUT alone or combined with ImmTOR. These mice were then followed for a year after treatment initiation (a middle-aged mouse). As seen before, administration of AAV-MMUT was beneficial and resulted in a dramatic improvement in metabolic status, weight gain and survival.
A further decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), was observed after treatment with the AAV-MMUT was administered in combination with ImmTOR. This difference was even more profound after a second treatment, as the mice treated twice with AAV-MMUT + ImmTOR showed further reduction in pMMA while the mice treated twice with AAV-MMUT alone showed no further benefit. The mice treated with AAV-MMUT by itself developed high levels of antibodies against the AAV capsid after the first dose, which would be expected to inhibit the ability of AAV-MMUT to reach the liver during the second dose. In contrast, there were no anti-AAV antibodies detected after the initial treatment when AAV-MMUT was co-administered with ImmTOR. As a consequence, re-dosing of AAV-MMUT and ImmTOR provided additional benefit with further decrease in pMMA levels and increases in body weight. A similar trend was observed for a biochemical disease biomarker, fibroblast growth factor 21 (FGF21), which was earlier shown by Venditti’s lab to be an MMA disease correlate both in mouse models and human patients. In addition, there were higher numbers of the AAV-delivered MMUT gene in the liver and increased MMUT expression when ImmTOR was co-administered with AAV-MMUT.
In other words, this meant that more therapeutic AAV had entered liver cells and more MMUT enzyme was made, resulting in lower toxic metabolites and disease markers when AAV-MMUT was administered in combination with ImmTOR, especially after re-dosing. This was further confirmed at the end of the study when mouse tissues were analyzed. Although livers from all AAV-MMUT-treated mice showed a dramatic improvement compared to untreated MMA mice, this benefit was even more pronounced when AAV-MMUT was administered in combination with ImmTOR. Importantly, co-administration of AAV and ImmTOR was well tolerated by MMA mice with no adverse effects seen throughout the study. Collectively, it appears that combining of ImmTOR and AAV not only inhibits the antibody response against AAV capsid, but may also improve vector entry into target cells and enhance the expression of therapeutic MMUT gene in the mouse model of MMA.
These observations suggest that the combination of ImmTOR and hepatotropic AAV-MMUT gene therapy has the potential to provide therapeutic benefit to MMA patients and be superior to a conventional single-dose of AAV gene therapy thanks to two contributing factors. First, ImmTOR can limit the detrimental impact of anti-AAV antibodies on gene therapy with the goal of enabling re-dosing of AAV vector and second, it may increase vector entry into liver cells and elevate therapeutic gene production upon initial dosing. ImmTOR also has the potential to modify the inflammatory response in the liver to avoid long term daily immunosuppressive medications such as steroids that are commonly used in gene therapy trials. The safety and efficacy of the AAV vector combined with ImmTOR must be demonstrated in clinical trials, which we anticipate starting in early 2022.
This multi-pronged mechanism of ImmTOR action makes it an attractive candidate to enhance systemic gene therapeutic applications, particularly in those clinical indications where repeat vector dosing may be necessary. Liver-directed gene therapy for metabolic diseases such as MMA presents a strong case for ImmTOR co-administration. Early intervention (in children and even infants) is highly desirable to prevent disease progression, and the target organ is destined to grow substantially over time, resulting in vector dilution. The rapid and enhanced expression of the AAV delivered gene may enable therapeutic benefit at lower doses of AAV and faster onset of the therapeutic effects. Furthermore, mitigating the formation of anti-AAV antibodies by ImmTOR provides an ability to re-dose vector over time, maintaining or restoring the therapeutic benefit of the gene therapy.
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[1] Ilyinskii PO et al. ImmTOR nanoparticles enhance AAV transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia. Mol Ther Methods Clin Dev. 2021; 22:279-292. doi: 10.1016/j.omtm.2021.06.015.
[1] Chandler RJ, Venditti CP. Gene Therapy for Methylmalonic Acidemia: Past, Present, and Future. Hum Gene Ther. 2019; 30(10):1236-1244. doi: 10.1089/hum.2019.113.
[1] Ilyinskii PO et al. Enhancement of liver-directed transgene expression at initial and repeat doses of AAV vectors admixed with ImmTOR nanoparticles. Sci Adv. 2021; 7(9):eabd0321.
[1] Manoli I et al. FGF21 underlies a hormetic response to metabolic stress in methylmalonic acidemia. JCI Insight. 2018; 3(23):e124351.
From the Fall 2021 OAA Newsletter