University of Florida
Department of Pediatrics & Neuroscience

Author Of 3 Presentations

Disease Modifying Therapies – Mechanism of Action Poster Presentation

P0339 - Fingolimod immune suppression does not inhibit long-term efficacy of AAV gene immunotherapy   (ID 1914)

Speakers
Presentation Number
P0339
Presentation Topic
Disease Modifying Therapies – Mechanism of Action

Abstract

Background

While there is no cure for MS, current disease modifying therapies (DMTs) focus on some form of immune suppression to slow disease progression. To circumvent the need for long-term DMTs and their adverse side effects, our lab has developed a novel adeno associated virus (AAV) gene immunotherapy that not only selectively induces neuroantigen specific regulatory T cells (Tregs), but can also prevent and/or stop preexisting demyelinating disease in an animal model of MS. Thus, when considering the development of a clinical trial, it will be important to know if the patient’s current immune modulating DMT will interfere with the ability of AAV to induce Tregs or their function.

Objectives

To determine if the DMT drug fingolimod, which sequesters lymphocytes in lymph nodes, will interfere with or inhibit the efficacy of AAV gene-immunotherapy to induce antigen specific Tregs that are responsible for disease suppression/reversal and maintain tolerance.

Methods

Experimental Autoimmune Encephalomyelitis (EAE) was induce in C57BL/6 (B6) mice using MOG35-55 or in SJL mice using PLP139-151. In the B6 cohort, mice received an AAV immunotherapy vector expressing MOG and began receiving oral gavage of fingolimod at the first sign of tail paralysis (~10 days post EAE) and daily for 25 days. In the relapsing-remitting SJL model, mice began receiving daily oral fingolimod during the first remission ~15 days after EAE induction. Ten days later, mice received an AAV vector expressing PLP. At ~35 days post EAE, fingolimod treatment was suspended. Disease severity was recorded daily on a standard 5-point scale of neurological disability.

Results

In the B6 experiment, mice that received both immunotherapy and fingolimod quickly reducing disease symptoms and remained essential disease free after fingolimod was discontinued, until end of experiment @45 days. In contrast, control mice receiving only fingolimod, relapsed shortly after fingolimod was discontinued and by day 35 disease severity was equal to EAE only controls.

In the SJL experiment, mice receiving AAV immunotherapy and fingolimod or fingolimod only failed to relapse during treatment. However, once fingolimod was withdrawn, control mice quickly relapsed into severe disease. Whereas, mice that also received the immunotherapy remained disease free, until end of experiment @65 days post EAE.

Conclusions

The data clearly suggests that simultaneously or overlapping treatment using the DMT, fingolimod, shows no apparent inhibition on the long-term effectiveness of the gene immunotherapy vector. In fact, the combinatorial effect may increase the effectiveness. In sum, our AAV gene-immunotherapy has significant clinical relevance as it restores a persistent and continuous immune tolerance such that long-term continuous DMT may be unnecessary for MS patients. Furthermore, when considering patient selection for clinical trials, patients currently taking fingolimod may not have to excluded.

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Experimental Models Poster Presentation

P0938 - An effective gene-immunotherapy for Multiple Sclerosis in mice. (ID 1444)

Speakers
Presentation Number
P0938
Presentation Topic
Experimental Models

Abstract

Background

Autoimmune disease like MS present with a loss of tolerance against endogenous proteins. Regulatory T cells (Tregs) maintain homeostasis between immune activation and suppression; however, in MS the mechanisms dependent on thymically produced Treg cells appear insufficient to prevent the initial priming of encephalitogenic T cells. Using an animal model of MS (experimental autoimmune encephalomyelitis (EAE)), we have shown that neuroinflammation and clinical symptoms can be prevented or even completely ameliorated by extrathymically induced antigen specific Tregs following tolerogenic gene immunotherapy. Historically, antigen specific therapies have remained elusive due to the myriad of known and unknown encephalitogenic epitopes present in patients due to differing HLA/MHC backgrounds. Developing a treatment capable of overcoming these hurdles may provide the most effective option for patients suffering from MS.

Objectives

Demonstrate the efficacy and dynamic ability of a novel Adeno-Associated Virus (AAV) gene immunotherapy to not only prevent disease onset, but to also ameliorate 'reverse' preexisting demyelinating EAE disease: 1) induced with various immunodominant and non-dominant encephalogenic myelin oligodendrocyte glycoprotein (MOG) antigens and, 2) do so in multiple strains of mice that have different HLA/MHC genetic backgrounds. Overall, validating the ability of the vector to be an effective therapy without prior knowledge of antigens or genetic background.

Methods

Demyelinating EAE disease was induced in genetically diverse mice (C57BL/6, H2b; DBA/1, H2q; SJL/J, H2s) using combinations of immunogenic epitopes (MOG35-55, MOG79-96, MOG92-106, or MOG1-125) emulsified in adjuvant. For Prevention, mice were given a single dose of a hepatocyte directed AAV vector expressing MOG that induces antigen specific Tregs and restore immune tolerance or control vector, 2-weeks prior to inducing EAE. For Reversal of preexisting disease, the therapeutic vector was given after disease onset as mice reached various predetermined levels of disease severity. Mice were monitored daily for changes in clinical score.

Results

For Prevention: Mice receiving the therapeutic vector showed no signs of disease onset, cellular infiltration or demyelination within the spinal cord. In contrast, controls developed inflammation and severe demyelinating EAE. For Reversal: Treated mice had lower disease peaks and a significant reduction in neurological impairment (clinical score returned to near baseline) with virtually no cellular infiltration or demyelination, compared to controls.

Conclusions

We have developed and demonstrated efficacy of a powerful AAV gene immunotherapy capable of dynamically adjusting to the unique antigen requirements needed to restore tolerance and independent of genetic background. This gene immunotherapy has the potential to be a paradigm shifting treatment options for MS.

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Experimental Models Poster Presentation

P0989 - PLP gene immunotherapy ameliorates disease and prevents epitope spreading in mouse models of RRMS (ID 1860)

Speakers
Presentation Number
P0989
Presentation Topic
Experimental Models

Abstract

Background

We recently developed a gene immunotherapy that reverses paralysis in a murine model of MS. However, the efficacy has only been established in the primary progressive disease model. Whereas, proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice is an animal model that resembles human disease with an initial relapsing- remitting (RR) disease phase followed by a secondary progressive stage. Given that ~85% of MS patients are diagnosed with RRMS, demonstrating the therapeutic efficacy in models that better resemble clinical disease an essential step toward a cure.

Objectives

Demonstrate the therapeutic potential of Adeno-Associated Virus (AAV) gene immunotherapy to prevent and/or reverse disease in multiple models of RRMS. Additionally, to show that this novel immunotherapy is also effective at abrogating epitope spreading in preexisting disease.

Methods

A single hepatocyte directed AAV vector expressing mouse PLP was engineered to induce/restore antigen-specific tolerance to multiple antigenic PLP epitopes. EAE was induced in SJL (H-2s) and C57BL (H-2b) mice using PLP139-151 or PLP178‑191 in adjuvant. For prevention of RR-disease, a single dose of therapeutic (or Null control) vector was given >7 days prior to EAE induction. For therapeutic reversal of pre-existing disease, immunotherapy treatment was administered after the first appearance of symptoms (early reversal) or during the first remission (late reversal). To demonstrate control of epitope spreading, PLP178‑191 was used to induced disease in SJL mice.

Results

For prevention of RR disease: therapeutic vector completely blocked clinical disease, cellular infiltration, and demyelination in PLP139-151 immunized SJL and C57BL/6 mice. For reversal RR disease: by day 30 of RR-EAE, AAV.PLP gene immunotherapy abrogated disease, reduced cellular infiltration, and suppressed demyelination, in both early and late treated mice. To evaluate efficacy against epitope spreading, cohorts of SJL mice were immunized with PLP178-191, an de novo epitope known to be revealed after induction with PLP139-151. AAV.PLP completely resolved disease in >90% of treated mice, rapidly after an initial but mild onset. In contrast, 100% of the control mice relapsed after the initial remission phase.

Conclusions

For the first time we provide definitive evidence that AAV directed gene immunotherapy efficiently prevents the development of disease as well as abrogates pre-existing disease and relapses in genetically different murine models of RRMS. The data further supports that treatment dynamically adjusts to protect against epitope spreading. Clinical translation of this novel gene immunotherapy could result in therapy that could effectively prevent or minimize the early inflammatory responses associated with MS, thus stopping the transition into secondary progressive MS and significantly improving the quality of life for MS patients.

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Presenter Of 1 Presentation

Experimental Models Poster Presentation

P0989 - PLP gene immunotherapy ameliorates disease and prevents epitope spreading in mouse models of RRMS (ID 1860)

Speakers
Presentation Number
P0989
Presentation Topic
Experimental Models

Abstract

Background

We recently developed a gene immunotherapy that reverses paralysis in a murine model of MS. However, the efficacy has only been established in the primary progressive disease model. Whereas, proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice is an animal model that resembles human disease with an initial relapsing- remitting (RR) disease phase followed by a secondary progressive stage. Given that ~85% of MS patients are diagnosed with RRMS, demonstrating the therapeutic efficacy in models that better resemble clinical disease an essential step toward a cure.

Objectives

Demonstrate the therapeutic potential of Adeno-Associated Virus (AAV) gene immunotherapy to prevent and/or reverse disease in multiple models of RRMS. Additionally, to show that this novel immunotherapy is also effective at abrogating epitope spreading in preexisting disease.

Methods

A single hepatocyte directed AAV vector expressing mouse PLP was engineered to induce/restore antigen-specific tolerance to multiple antigenic PLP epitopes. EAE was induced in SJL (H-2s) and C57BL (H-2b) mice using PLP139-151 or PLP178‑191 in adjuvant. For prevention of RR-disease, a single dose of therapeutic (or Null control) vector was given >7 days prior to EAE induction. For therapeutic reversal of pre-existing disease, immunotherapy treatment was administered after the first appearance of symptoms (early reversal) or during the first remission (late reversal). To demonstrate control of epitope spreading, PLP178‑191 was used to induced disease in SJL mice.

Results

For prevention of RR disease: therapeutic vector completely blocked clinical disease, cellular infiltration, and demyelination in PLP139-151 immunized SJL and C57BL/6 mice. For reversal RR disease: by day 30 of RR-EAE, AAV.PLP gene immunotherapy abrogated disease, reduced cellular infiltration, and suppressed demyelination, in both early and late treated mice. To evaluate efficacy against epitope spreading, cohorts of SJL mice were immunized with PLP178-191, an de novo epitope known to be revealed after induction with PLP139-151. AAV.PLP completely resolved disease in >90% of treated mice, rapidly after an initial but mild onset. In contrast, 100% of the control mice relapsed after the initial remission phase.

Conclusions

For the first time we provide definitive evidence that AAV directed gene immunotherapy efficiently prevents the development of disease as well as abrogates pre-existing disease and relapses in genetically different murine models of RRMS. The data further supports that treatment dynamically adjusts to protect against epitope spreading. Clinical translation of this novel gene immunotherapy could result in therapy that could effectively prevent or minimize the early inflammatory responses associated with MS, thus stopping the transition into secondary progressive MS and significantly improving the quality of life for MS patients.

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