Combination Therapy Is the New Gene Therapy?

Main Text

Multiple sclerosis (MS) is a complex inflammatory, demyelinating disease with a characteristic relapsing and remitting pattern. Although the pathogenesis of the disease is still controversial, the presence of CD4⁺ and CD8⁺ T cell infiltrates and the identification of oligoclonal immunoglobulins in the cerebrospinal fluid of patients strongly suggest that MS is an immune system disorder involving different antigens of the nervous system and, therefore, is ultimately classified as an autoimmune disease. In this issue of Molecular Therapy, Keeler and colleagues¹ use an established model of MS to study the dominant role of peripheral tolerance induced by adeno-associated virus (AAV) liver gene therapy in preventing the disease onset. The most important goal of gene therapy for autoimmunity is prevention, and Keeler and colleagues provide evidence of effective and stable remission of the disease when treated in an advanced state using liver gene therapy and rapamycin, a US Food and Drug Administration (FDA)-approved drug used in transplants. The demonstration of a strong synergy between rapamycin and AAV-mediated liver gene transfer in the control of established autoimmune responses opens the way to several possible translational applications.

Experimental autoimmune encephalitis (EAE) is the most commonly used model of MS and mimics both the key pathological features of MS and the remyelination process characteristic of the remitting phase of the disease. The EAE model is obtained via immunization of animals with an antigen derived from a myelin protein (myelin oligodendrocyte glycoprotein [MOG]) formulated in complete Freund’s adjuvant, a potent immune system booster. Immune cells primed against MOG then migrate to the target tissue, where they recognize the auto-antigen and initiate the inflammatory process. The use of EAE as a model for MS allowed for the development of a variety of immunomodulatory strategies that have entered clinical practice and improved MS treatment and the quality of life of patients.² However, these treatments are somewhat limited in efficacy and results in chronic, global immunosuppression, which is not risk-free. Thus, although challenging, re-establishing peripheral tolerance in an antigen-specific manner is a highly attractive therapeutic approach to MS.

AAV vectors are effective and safe tools for in vivo liver transduction as demonstrated in both preclinical animal models and clinical trials.³ Induction of robust transgene-specific tolerance by AAV-mediated liver gene transfer has been documented in several studies and is dependent on the induction of regulatory T cells (Tregs) that actively suppress the anti-transgene immune responses.⁴ Rapamycin is a drug known to promote the stabilization of Treg phenotype, as it has been shown to promote tolerance to protein replacement therapy in hemophilia A models,⁵ and Treg expansion in vitro and in vivo.⁶ Therapeutic transplantation of autologous in vitro-expanded Tregs has been used to treat MS and other autoimmune diseases in animal models. However, the clinical translation of these results demonstrated only transient efficacy, possibly due to decreased survival or loss of the suppressive phenotype of the transplanted Tregs.⁷

In their new study, Keeler and colleagues¹⁷ showed that liver-directed gene therapy with an AAV8 vector expressing MOG, under the transcriptional control of a potent liver-specific promoter, resulted in the induction of transgene-specific tolerance. This suppressed anti-MOG immune responses and ameliorated the neurological impairment associated with EAE in mice. AAV8-MOG administration controlled the immune response even when the vector was given after EAE induction in mice with a low mean clinical score (MCS), corresponding to the early phases of the disease. In mice with an advanced EAE phenotype, the combination of AAV8-MOG and rapamycin showed a near complete remission of the EAE by day 30.

Long-lasting therapeutic liver gene transfer was first achieved in a clinical trial of gene therapy for hemophilia B led by Dr. Nathwani and colleagues.⁸ The treatment of hemophilia B patients, based on repeated injections of human coagulation factor IX (hFIX) results in the formation of anti-hFIX inhibitor antibodies only in a small percentage of patients (1.5%–3%⁹). The exclusion of patients that developed inhibitory antibodies in response to hFIX protein infusion in this clinical trial prevented a careful evaluation of any eventual tolerance induced by transgene expression in the liver. Nevertheless, the extensive experience in liver transplants¹⁰ and the mechanisms leading to the persistence of hepatitis B virus in the liver during chronic infections¹¹ highlight the pro-tolerogenic potential of this tissue in humans. This, together with the demonstration of the induction of a robust tolerance toward the transgene in large animal models after AAV-mediated liver gene therapy,¹² supports the idea that liver tolerance is mediated by a universal mechanism across species.

Liver gene therapy has been proposed as an immunomodulatory strategy in several settings,12, 13 including autoimmunity.¹⁴ This approach could potentially reach the clinic in the near future and provide proof of concept of the safety and efficacy of liver tolerance as a treatment for human disease. As such, the tolerization strategy proposed by Keeler and colleagues¹⁷ is relatively simple and has a strong translational potential. A careful definition of the mechanism(s) leading to such a powerful effect on the EAE is important. In particular, a critical point is the identification of the site where the expansion of Tregs occurs following the administration of liver gene therapy and rapamycin. To this end, two different models could be proposed. First, Tregs induced in the periphery migrated in the CNS, where they encountered the antigen and expanded through the action of rapamycin. Second, Tregs are both induced and expanded in the periphery and then migrated in the CNS. This will be particularly relevant considering the peculiar characteristics of Tregs identified in the CNS15, 16 and could explain the synergy between gene therapy and rapamycin. The role of the liver in the maintenance of Treg population should also be clarified, and, eventually, the effects of long-lasting ectopic expression of MOG in hepatocytes should be evaluated. Other important questions remain to be answered before translation of this therapeutic approach to MS patients. Although EAE is a very robust and reliable model of MS, it carries the limitation that anti-MOG-specific immune responses are seen in MS patients, but the description of multiple antigens and the inconsistency of anti-MOG antibody response in patients indicates that, in humans, the activation of the immune system may be more complex than in mouse models.¹⁷ This indicates that, although promising, the future translation of an AAV-based gene therapy for immunomodulation in MS may require a comprehensive identification of the antigens that sustain the neuroinflammatory process underlying the disease.

AAV liver gene transfer in combination with rapamycin is bringing us one step closer to treating autoimmunity. While this is a potential breakthrough in the treatment of MS, the work of Keeler and colleagues¹ inherently expands the clinical applications of AAV vectors and paves the way to the future of gene therapies combined with conventional drugs to achieve previously unthinkable results.