Immune considerations relevant to DMD and dystrophin ......Activates complement to create a...
Transcript of Immune considerations relevant to DMD and dystrophin ......Activates complement to create a...
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M. Carrie Miceli, Ph.D.Professor of Microbiology, Immunology and Molecular Genetics
Co-Director, Center for Duchenne Muscular DystrophyDavid Geffen School of Medicine and College of Letters and Sciences at UCLA
277B Biomedical Sciences Research Building
Immune considerations relevant to DMD and dystrophin
replacement/correction therapies (not all immune responses are bad)
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Basic Immunology: Self Non-Self Discrimination for Defense,Self-Tolerance, and Regeneration
Innate Immune Response Adaptive Immune Response high specificity (viral AAV capsid or micro-dystrophin peptides)
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Aggressive(Cytotoxic Lymphocytes CTL)
Tolerizing
B cellantibodies viral neutralizing
Memory
(Treg + others)
RegenerativeTreg, M2 + others
-1st line of defense Looks for Danger Associated Molecular Pattern=DAMPS
bacterial sugars structureViral capsid, viral DNA/RNAStressed or dying cells
-Alerts Adaptive ImmuneCells to DANGER-
T cells
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Macrophagedepletion
. Immune response to muscle damage guides regeneration
Acute injury
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Activates complement to create a fibrin/platelet patch (clot) at the lesion site.
Clear debris, pro-inflammatory cytokinescells
Repair and resolution
In response to acute injury waves of infiltrating cells coordinate patching , stem cell activation, muscle repair
In DMD , Chronic damage Asynchronous repair
Improper resolutionIneffective regeneration Profibrotic
Can we reset?-
Can we intervene with drugs-antifibrotics or immune modulators-dystrophin replacement
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In Duchenne Muscular Dystrophy chronic injury prevents resolution or immune response, drives muscle damage and fibrosis
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Dalia Burzyn , Wilson Kuswanto , Dmitriy Kolodin , Jennifer L. Shadrach , Massimiliano Cerletti , Young Jang … Diane Mathis...
A Special Population of Regulatory T Cells Potentiates Muscle Repair and Inhibits Fibrosis
Cell, Volume 155, Issue 6, 2013, 1282 - 1295
T-Reg’s Suppress specific inflammatory Immune responsesBlocks fibrosis (IL-10)Promote muscle regeneration (amphiregulain)
Upregulation improves mdx DMD mouseDownregulation worsens
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Can immune modifiers limit fibrosis/promote regeneration?
Rosenburg and Woodcock, Nature Immunology
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Dystrophin replacement strategiesself/non-self discrimination
• Will there be an immune response that limits safety or efficacy?• -exon skipping, NS read-through and micro-dystrophin gene therapy all
strive to make an altered dystrophin protein in boys who lack dystrophin. • Will this dystrophin proteins be seen as non-self threat?
• Micro-dystrophin gene therapy has additional potential immune challenge to AAV vector • Can we induce specific self-tolerance to AAV/dystrophin?• Will dystrophin replacement prevent/reverse immune pathology?
• Reverse tissue damage and fibrosis, while promoting regeneration
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∆3990 ∆R4-23/∆C µDys5R 9SOLID
Gene therapy for DMD
Sarepta*slide courtesy of Dongsheng Duan
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Mingozzi and High Annual Review of Virology 2017
We don’t know if there will be a immune response to AAV micro-dystrophin gene therapies that limits/tempers efficacy or safety in AAV sero-negative DMD patients? Complement activation?
Why are AAV seropositive boys currently excluded from trials?
Immune reponse to AAV Gene Therapy
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Exposure to AAV in the wild induces production of AAV specific neutralizing antibodies that can block GT delivery
80% neutralizing
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Immune Response to AAV: pre-existing antibodies
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Potential Solutions for Pre-formed Antibodies
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Muscle in DMD is not “normal”
Chronic Immune Activation upregulation of class I MHCclass II MHCTLR7cytokines
Pre-existing AAV or dystrophin reactive T cells in DMD?
DMD- Intra-Muscular GT Injection(or no expression)
Screen for and excludeindividuals with
pre-existing AAV or dystrophin reactive T cells (g-IFN)
immunosuppress
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(AAV2 and AAV8)
NOT tested in DMD
A unique moDendritic cell population identified which produces IL-6 and IL-1b ; blockingIL-1b with antibodies prevented AAV antibody production.
Deep immune profiling
JCI 2018
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Immune Response to AAV: Innate Immune Response
Mingozzi et al
TRL2-AAV capsidTRL9-AAV vector/na
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Pro-inflammatory Cytokines/ChemokinesIL-6 and IL-1b and others
PhagocytesComplement Activation
Upregulation of T cell co-stimulators on antigen presenting cells
DAMPS-Danger associated molecular pattern receptors 1st line of defense; alert adaptive responseCan we identify players and modulate?
Broad exposure to DANGER=Capsid proteinsssDNAdsDNABacterial sugarsCell stress Cell damage
Candidate AAV GT triggers:AAV capsidTLR2 AAV vector/ssDNATLR9
In DMDTRL7 ssRNA
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Adaptive immunity: B cells and T cells each have surface receptors that recognize specific antigens which trigger the activatand development of immune effectors and memory cells.
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Diverse CD4 and CD8 T cells subsets regulate immune activation and self tolerance
Subset distinguished by co-expression of surface antigensand functional output; plasticity and intermediates observed
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Immune Response to AAV: Adaptive Immunity Specific for AAV-vector or for dystrophin transgene?)
Micro-dystrophinor AAV capsid
Y
CD4
CD8CTL
CD8CTL
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APC
APC CD4
(CD8 helpers)(Tregs and others)
CD4 T cells (B helpers)
CD8CTL
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AAV Immune Response Dystrophin Immune Response
Potential Problems and Solutions
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Impact of Immune-Modulatory Drugs on Regulatory T CellFurukawa, Wisel, MD, and Tang, Transplantation 2016;100: 2288–2300)
All T/B cell responses
Vs novel mechanism/drugs for inducing antigen specific tolerance.
The goal of T cell immunosuppression for gene therapyis to block Teff and induce tolerance (Tregs + other).
(rapamycin)
Can we borrow from fields of transplantation or autoimmunity where the goal is also to dampen inflammation and tolerize?
Barry B
Tacromilus/rituximab (rapamycin)
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Front. Immunol., 09 November 2015 | https://doi.org/10.3389/fimmu.2015.00569
T cell activation versus tolerance dictated by:
(generalizations)
• Status of the APC and local cytokines
• Upregulation of inhibitory proteins on the T cell surface
• Low level chronic exposure to antigen often tolerogenic
CTLA4 or PD1
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Ongoing approaches to induce antigen specific tolerance
J Exp Med16 January, 2019 http://doi.org/10.1084/jem.20182287
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Is pre-exposure to dystrophin inconsequential,tolerizing, or activating?• Revertant fibers? • Exon skipping pre-treatment?• Ataluren pretreatment?
• Priming (vaccination)• Tolerance?
• Low levels of chronic activation can lead to clonal T cell exhaustion/anergy• Combination therapy?
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• Co-administration of rapamycin nanoparticles with AAV prevents activation of AAV specific B and T cell and induction of memory responses in mice and non-human primates (Not tested in DMD).
• Likely through induction of antigen specific Tregulatory cells
Nature Communications 2018
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Cas9 is a bacterial protein; pre-formed antibody T cell immunity immunity blocking efficacy
0% had T cells specific for Cas965% had antibodies specific for Cas9
96% had Cas9 specific T cells85% had antibodies specific for Cas9
Exclude patients with preformed immunity? Immunosuppress? Identify Cas9-like protein with lo/no immunogeneicity.
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• Muscle biopsy- limited number• how many; and when• Needle biopsies vs open muscle biopsy (infiltrate and
regeneration evaluation)
• MRI/MRS- Imaging DMD • Peripheral blood –
• Standards for human immune monitoring of subsets evolving with improved ability to characterize subpopulations and functionality
• Can detect AAV/dystrophin reactive T cells in blood• Can better characterize T cell subsets using multi-parameters
• Deep immune profiling using CyTOF and single cell RNAseq• Perhaps a signature can serve as a biomarker for efficacy or tolerance
How do we monitor response?
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Disclosures: Myself or a member of my family has received compensation and/or travel from the above.
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Adaptive Immunity:First exposure activates B and T cells for defense and memories that can respond faster and better upon re-
dosing
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Natural AAV infection AAV-GT infection AAV-GT infection Natural AAV infection AAV-GT re-infection
(high dose) AAV-GT infection
(High dose) AAV-GT re-infection AAV-GT infection
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B cells T cells