Master Immunotechnologies et Biothérapies de l'UPMC

Immunological problems in gene therapy:

Immune responses against the vectors and the transgenes and possible strategies tothe transgenes and possible strategies to 

control this immune response.

Anne Galy PhDInserm U951, UMRS_951 UEVE/EPHE

Genethon, Evry  

Principles of Immune ResponsesPrinciples of Immune Responses

Natural Immunity(non specific, innate)

Adaptive Immunity(specific)

- Natural barriers

Inflammatory reactions

- Humoral immunity (Ig)

Cellular immunity- Inflammatory reactions

- Complement factors

- Cellular immunity

1. Recognition

2. Activation

3. Effector phase

4. Memory

Cooperation

Principles of Immune ResponsesPrinciples of Immune Responses

GTV     

Adapted from Gregersen and Behrens Nature Reviews Genetics 2006

Principles of Immune ResponsesPrinciples of Immune Responses

Anatomical and Physiological considerationsBarriers Sanctuaries FiltersBarriers – Sanctuaries ‐ Filters

Natural barriers FlowSkinBlood‐Brain barrierBlood vessels

Adhesion molecules

Immune sanctuariesCNS eye

Extracellular components

Specialized cells

FiltersLiverS lSpleenKidneys

Gene Therapy: ConceptsGene Therapy: Concepts

Gene Therapy relies on the introduction of geneticsequences in cells, tissues or organisms with the objective f i d i i l i iof inducing, correcting or regulating gene expression

to obtain a therapeutic effect.

Gene therapy is tested and used for various applications in:

Monogenic diseases CCancerComplex conditions such as cardiovascular conditionsInfectious diseasesInfectious diseases

Gene Therapy: ConceptsGene Therapy: Concepts

lHigh specificity

Low toxicity

Delivery TropismBiodistribution

Low toxicity

FeasibilityImmune responses

ProductionCurrently:HSCSkin cells

Production

CurrentlyEye

adapted from NIH

EyeLiverBrainMuscleMuscle

Adapted NIH source

Gene Therapy : Tools

1‐ NAKED DNAIntroduced by intramuscular route or with a « gene gun »  Plasmid DNA containing sequences necessary for the expression of a given proteinPlasmid DNA containing sequences necessary for the expression of a given protein 

* Not very effective for gene expression purposes but useful for vaccine strategies   

2‐MOLECULAR COMPLEXES AND LIPOFECTION2‐MOLECULAR COMPLEXES AND LIPOFECTIONNon‐viral vector systems that augment the passage of DNA through the membrane.  Since DNA and cell membranes are negatively charged,  polylysines or cationic lipids are added to neutralize these charges and to augment the  binding of the complex to the cell membrane.   Several other components are added to improve the efficiency of gene transfert for instance:Several other components are added to improve the efficiency of gene transfert, for instance:  ‐ proteins like transferin which improves gene transfer in endocytic vesicles.   ‐ viral (fusogenic) envelope proteins to improve the transfer of DNA in the cytosol.  ‐chloroquine in order to destabilize endosomes, burst the vesicles to liberate the gene more effectively.  

* Moderately efficient but attractive for chemistry, pharmacologic and « galenic »  

3‐ VIRAL VECTORSIntroduced in the cell to transport genetic material Transcription is performed by hijacking the cellularIntroduced in the cell to transport genetic material. Transcription is performed by hijacking the cellular metabolism.  The basic principle of recombinant vector technology is to remove certain sequences from the viral genome to render it safe, and to introduce the transgene of interest in this recombinant vector for delivery.  DNA viruses used are: adenovirus AAV SV40 herpes simplex virus vaccinia virusDNA viruses used are:  adenovirus, AAV, SV40, herpes simplex virus, vaccinia virus  RNA viruses  are:   retrovirus (RV), polio virus or inflluenza virus. 

* Viral vectors are efficacious methods for gene transfer 

Gene Therapy: ApplicationsGene Therapy: Applications

Gene Therapy can be performed with various protocols (in vivo ex vivo various cells various agents various routes)vivo, ex vivo, various cells, various agents, various routes)

Gene Therapy may have diverse applicationspy y ppMonogenic diseasesCancerComplex conditions such as cardiovascular conditionsInfectious diseases

Immune responses induced by gene therapy may be eitherunwanted (i.e. treatment of genetic deficiencies) or ( g )desired (cancer immunotherapy).

UnwantedImmune responses in GT Applications

Adeno‐associated virus

Single stranded 4700pb DNA genome

Non enveloped defective parvovirus

Icosahedral capsid : VP1 VP2 & VP3 proteins ‐> ratio 1:1:10

recombinant AAV vectors

AAV d 81 h li i l i l (j l f di irAAV vector used 81 gene therapy clinical trials (journal of gene medicine, update June 2011)

Absence of pathogenicity of AAV wt virus in humans

Gene transfer efficient in vivo in well differentiated tissues

Stable and long term expression of the transgene

rAAV genome is maintained as multimeric episomal formsg p

Immunogenicity of rAAV : considered as a limitation based on human clinical data

Clinical trials with rAAV (as of 2008)

Abs to capsid

Abs not neutralizing

rAAV vectors are used in > 50 gene therapy clinical trials

NAB

ptrials.

Over 500 subjects have received AAV, mainly AAV2, 

b t f h b NABNAB, CTLs anti capsid

but very few have been treated systematically or analyzed for immune responses. 

NABImmune responsesto AAV capsid

Recent concern.

adapted from C. Mueller and T. Flotte H Gene Ther 15: 858, 2008

Evidence of Cellular Immune Responses to rAAV capsid in man

Mingozzi et al. Nature Medicine 2007

Manno et al. Nature Medicine 2006

2007

For more recent data see: Immune responses to AAV in clinical trials by F. Mingozzi and K. High, Curr Gene Ther. 2011 Aug1;11(4):321-30.1;11(4):321 30.

INNATE IMMUNERESPONSES ?

WHY DO WE CARE ABOUTTHAT IN GENETHERAPY ??

Jesse Gelsinger

Died 98 hours after infusion of Ad5 for OCT deficiencyMassive innate immune response

First death due to gene therapyHas changed the field : Informed consents, preclinical evaluation GCPNo longer overly‐optimistic vision of GT : Immune responses to vectorNo longer overly optimistic vision of GT : Immune responses to vector

UnwantedImmune responses in GT Applications

Immune responses against the Vector components (capsid, ADN)

Can be very violent (innate inflammatory Responses)‐ Can be very violent (innate inflammatory Responses)

‐Can be pre‐existing‐Prevent Vector re‐administration (neutralizing antibodies)Prevent Vector re administration (neutralizing antibodies)‐Memory effect

‐Contribute to immune responses against gene‐modified cells leading toContribute to immune responses against gene modified cells leading to ‐Toxicity and tissue destruction‐Loss of therapeutic efficacy

Immune responses against the transgene

‐Can lead to loss of gene‐modified cells‐Can lead to toxicity‐Can lead to loss of therapeutic activity

Important Parametersp

Important Parametersp

‐Nature of the Gene Therapy Vector

‐Dose

‐Route of administration

P it d f t i t dj t‐Purity and presence of contaminants or adjuvants

‐Existence of a tolerance or notExistence of a tolerance or not

‐Distinguish responses to Vector (not expressed but presented)or to Transgene (expressed and presented)

Recognition of, and response to vectortcomponents

rAAV

Adapted from Gregersen and Behrens Nature Reviews Genetics 2006

rAAV, Cytokines and Complement. Importance of in vivo context.  

AAV i fl I d i f ifl kirAAV vectors are « non‐inflammatory »... compared to Adenoviral vectors.

Induction of pro‐iflammatory cytokinesin macrophages in vitro is only visible in the presence of complement.

rAAV2

rAAV induces a transient inflammation which is dependent upon macrophages

C3

C3b

iC3b

NAB

 titers

NAB

 titers

Zaiss, Muruve et al. Journal of Virology 2002 Zaiss, Muruve et al. Journal of Virology 2008

C3i

How can rAAV possibly interact with innate receptors/sensors ? 

attachment

C3i

C3i

C3i

microtubulesclathrinvesicles

endocytosis

autoh

proteins ?

acidificationcapsid disassembly

lysosome

phagosome

late endosomeescape (PLA2)

proteasome

nls

P, Ub

nls

final capsiddisassembly

inflammasomecircularizationdsDNADNA repair machinery

inflammasome

DNA sensor ?

Activated adaptive immune responses ??

adpated from Pilchmair and Reis e Sousa Immunity 2007

Recognition of, and response to vectort

Unpublished experimental data:

components

Several pathogen and inflammation molecules were tested among which:•TLR9 = an endosomal receptor for CpG DNA. •MyD88 = an adaptor for several innate/inflammation pathways.

h d d hMice with gene‐targeted mutations in TLR9 or MyD88 were injected with rAAV.AAV‐specific IgG are measured in their blood.Results show that :TLR9 is not essential for the ability to produce anti‐AAV1 IgG, but MyD88 is critically required.

1E+06TLR9-/-

ttite

rs)

1E+05

IgG

(end

poin

t

1E+03

1E+04

AV1-

spec

ific

1E+02Ant

i-AA

NOTE:     T cell development controls  IgG switch

Th cytokines determine isotype switching

TGF

Mice produce TH1‐dependent antibodies to rAAV1  (IgG2b and IgG2c)This critically requires MyD88This critically requires MyD88 

TLR9 is involved in controlling the  balance IgG1/IgG2c 

0,8

1

IgG1 IgG2b IgG2c IgG3

MyD88-/-wt

0,6

0,8

1,41,61,8

1 41,61,8

2

0,2

0,4

0,6**

y

0,2

0,4

0 20,40,60,8

11,2

***0 20,40,60,8

11,21,4

***

1,2

01E+2 1E+3 1E+4 1E+5

V1 E

LISA

cal d

ensi

ty

1

***

01E+2 1E+3 1E+4 1E+5

00,2

1E+2 1E+3 1E+4 1E+5

1 61,8

00,2

1E+2 1E+3 1E+4 1E+5

1 82

0,4

0,6

0,8

1TLR9-/-wtA

AVO

ptic

0,4

0,6

0,8 ***

0,60,8

11,21,41,6

0 60,8

11,21,41,61,8

*

0

0,2

0,4

1E+2 1E+3 1E+4 1E+50

0,2

1E+2 1E+3 1E+4 1E+50

0,20,40,6

1E+2 1E+3 1E+4 1E+50

0,20,40,6

1E+2 1E+3 1E+4 1E+5

Inverse dilutions of sera

Tools to study specific T cell responses

AAV2/1

DBY NAGFNSNRANSSRS

DBY-AAV2/1

VP1

VP2

VP3

34

Cap

DBY = class II epitope  CD4+ T cells

Transgene-HY

Injection

± targeted

CD8+ MataHari anti-UtyCD4+ Marilyn anti-DBYCD4 F P3GFP/GFP ti DBY peptide or

expression

CD4+ FoxP3GFP/GFP anti-DBY

Immunologic tools

peptide orpentamer

g

rAAV induces capsid‐specific Th1 T cells in MyD88‐dependent manner (left). The priming of capsid‐specific Th1 T cells depends on MyD88 (right).

FACS

Dby

6ce

lls **

100*

100*

70

80

CD

4+

0 010

**

Dby

U p

er

10

6

60

80

60

80

40

50

60

70

+C

D4+

/ 0.010

FN-

SFU

20

40

20

40

10

20

30

CD

45.

1+ 0.005

IF

PBSwt

DBY-rAAV1

wt

DBY-rAAV1

MyD88-/-

00 PBS

wtDBY-

rAAV1wt

DBY-rAAV1

MyD88-/-‐20

00 1 4 ‐10 rAAV1

wtDBY-

rAAV1wt

DBY-rAAV1

MyD88-/-

0.000

y y y

Summary: response to vector componentsy p p

-rAAV induces strong antibody responses, characterized by A Th1 antibody profile (IgG2c and IgG2b in C57Bl/6 mice)

W fi d th t AAV i d id ifi Th1 CD4 T ll i i hi h-We confirmed that rAAV induces capsid-specific Th1 CD4 T cells in mice whichExplains not only the strong IgG response but explains the potential to obtainPathogenic capsid-specific T cell responses in certain models.

-MyD88 is critically required for the antibody response to rAAV and also for The development of capsid-specific Th1 T cells.

- MyD88 is an essential molecular target.

-Studies on mechanisms related to antigenic presentation T cell priming andStudies on mechanisms related to antigenic presentation, T cell priming and role of MyD88 in various subpopulations of antigen-presenting cells andidentification of MyD88-related pathways is currently ongoing.

Transgene‐specific immune responses  

AAV-CMV-tansgene

DC

DCtissueT CD4

T CD8

DCtissue

Lymphoid organs

Potential destruction Of gene-modifed cellsLoss of therapeutic activity

Immune response to a transgene in a gene therapy model 

AAV2/1-mediated gene transfer of alpha-sarcoglycan in a model of LGM2D

Sgca-KO mice

AAV2/1

g

Immune responses to transgenes lead to:u e espo ses to t a sge es ead to

-Inflammation-loss of therapeutic efficacy-production of anti-transgene antibodies

anti-Sgcap g

Tissue-specific expression systems improve

anti-CD8

Tissue specific expression systems improveand point at transgene antigenic presentationbeing a main problem

Fougerousse, Bartoli et al., Mol Ther 2007

Micro ARNs (miRNAs)

Lodish, Zhou, Liu and Chen. Nature 2008

Micro RNA regulated expression of the transgene 

mir142.3p expressing cell mir142.3p negative cell

Hematopoietic cell Non-hematopoietic ll

mir142-3p targets mir142-3p targetsgenegene

mir142- 3p+ cellmir142- 3p-

Brown, Naldini et al., Nature Medicine 2006.

Chimeric SGCA‐HY transgene: Additional epitopes to follow immune response 

HY epitopeCMV D i

SV 40 polyAChimeric intron

Human α-sarcoglycan ITRITRCMV or Desminpromoter

AAV2/1

HY epitopeSV 40 polyA

Chimeric intron

ITR Human α-sarcoglycan ITRCMV promoter miR 142-

3pT

Immunologic tools

Injection

AAV_Sgca-HY

Sgca KO / C57Bl6

± targeting

CD8+ MataHari anti-UtyCD4+ Marilyn anti-DBYCD4+ FoxP3GFP/GFP anti-DBY peptide or

pentamer

g _

Mir142.3‐regulated transgene expression is sustained 

Vector Sgca-HY-mir142.3pT PBSSgca-HY

D14

Sgca-HY vectorSgca-HY-mir142.3pT vectorPBS

C57Bl6

Transgene‐specific immune responses  

Possibility to control immune responses to transgene by controlling Tg expressionPossibility to control immune responses to transgene by controlling Tg expressionTissue‐specific expressionUse of mir142.3p‐regulation in blocking CD4 and CD8 responses A useful manner to overcome a significant roadblock in many applications of rAAV GT

Li i ? T l ?Limits ? Tolerance ?

Control of unwanted immune responsesp

‐Minimize the risk‐Reduce immune system priming (tissue‐specific expression)‐Choose less immunogenic Vectors (nature of vector, serotype, route)g ( , yp , )‐Reduce the risk of innate IR and adjuvants (purity, formulation)‐Reduce vector dose  increase vector specific activity‐Choose tolerant individuals/avoid gene‐null individualsg

‐Immunosuppression (not used universally)‐It works‐May be toxic (risk/benefit)‐Which drugs, how long ?

‐Preventative measures (still experimental)‐Plasmapheresis‐B cell depletion

Conclusions

Immune responses in GT can have serious and deleterious consequences

Immune responses to certain vectors seriously limit GT applications

GT vectors and GT products obey the same rules than other antigens or microbes

Immune responses induced by gene transfer are complex

Immune responses can be controlled but protocols remain arbitrary and controversial

I l di t t i t t l IR i GT d l i ti lIncluding strategies to control IR in GT models is essential

Not all GT applications have problems with IREx vivo treament of primary immune deficiencies‐ Ex vivo treament of primary immune deficiencies

‐ Vaccines, immunotherapy …

Non‐replicative recombinant particles are informative toolsNon‐replicative, recombinant particles are informative tools•To decipher some mechanisms of immune responses•To engineer new generations of vectors