Upload
jaya-sunil-lande
View
546
Download
0
Tags:
Embed Size (px)
Citation preview
Gene Gene therapytherapy ::fromfrom dreamdream to (to (toughtough) reality) reality
Serge Braun, AFM
GENE THERAPY :GENE THERAPY : Transfer of genes into cells to prevent or treat diseases
• Local or systemic administration• Hereditary or acquired diseases
An ideal gene therapy vectorAn ideal gene therapy vector
• Allows efficient and selective transduction of the target cells• The vector is maintained inside the cells• Expresses gene of interest at levels necessary for achieving therapeutic effects• Safe
Retrovirus/LentivirusAlphavirusMeaslesHerpes simplexAdenovirusAAVPoxvirus
Salmonella typhimuriumListeria monocytogenes
Plasmid (naked DNA)ElectroporationSonoporationGene gun
LipoplexesPeptidoplexesSolvoplexes
RNA transfer
VectorsVectors
Adenovirus Poxvirus
Non-viral
Retrovirus
Efficacy
Transientexpression
Vaccinesnon
chronic
No immune response
Repeated administr.
Low efficacy
Long term
+
-
+-
+
-+
Integration
Safety
chronicdiseases
+
-
Level ofexpression
Readministration ?
Targeting of different organs by viral vectorsTargeting of different organs by viral vectors
Lundstrom (2003) Trends in Biotechnology, 21: 117.
Construction of Vectors
E1A, E1B : transcription, transactivation, immortalization, transformation
E2A, E2B : viral DNA synthesis
E2A : transactivation of MLP
E4 : regulation of : DNA synthesis, early / late transcription, mRNA stability, splicing, apoptosis
E3 : immunomodulation : downregulation of MHC
pIX: capsid protein, transactivation
MLP (L1 - L5) : viral structural proteins, viral assembly
E a
r l y
L a
t e
ITR ITR
Ψ
MLP
L1L2
L3 L4 L5
E2B (pTP, POL)
E2A (DBP) E4
E3E1A E1B
3’
3’5’
5’pIX
IVa2
AdenovirusAdenovirus type 5 type 5 genomegenome
AdenovirusAdenovirus as as genegene transfertransfer vectorvectorLeft ITR Ad 5 genome E3°E1° Right ITR
Promoter Intron pATherapeutic Gene
Expression cassette
Production in a Production in a complementingcomplementing cellcell linelineLeft ITR Ad 5 genome E3°E1° Right ITR
Promoter Intron pATherapeutic Gene
E1/E3
Complementing cell line
Left ITR Ad 5 genome E3°E1° Right ITR
Promoter Intron pATherapeutic Gene
E1/E3
Complementing cell line
Production in a Production in a complementingcomplementing cellcell lineline
Left ITR Ad 5 genome E3°E1° Right ITR
Promoter Intron pATherapeutic Gene
E1/E3
Complementing cell line
Production in a Production in a complementingcomplementing cellcell lineline
Left ITR Ad 5 genome E3°E1° Right ITR
Promoter Intron pATherapeutic Gene
E1/E3
Complementing cell line
Production in a Production in a complementingcomplementing cellcell lineline
AAVAAV
Regulatory aspects:Regulatory aspects:
• GMPs: products of biological origin
• Containment and GMO’s: protect personnel and avoid dissemination: L3
• Conciliate GMPs and containment
Bioreactor, suspended cells
Large scale productionLarge scale productionof recombinant virusof recombinant virus
Clarification (Filtration)
Chemical elimination of enveloped viruses
Ion exchange chromatography Bioprocess apparatus
PurificationPurification
Buffers in disposablepallets tanks Molecular sifting
PurificationPurification
Raw After chromatography
Formulatedproduct
HPLC analysisHPLC analysis
ControlsControls throughoutthroughout the the processprocess
Final product
PurityAbsence of viral contaminants
PurityDosage of residuals
Identity, efficacy, stability
Rawmaterial
Cell, virusbanks
FERMENTATION
PURIFICATION
FORMULATION AND
REPARTITION
Identity, efficacy, stability
Bulk
Purified bulk
Quality control of the Final ProductQuality control of the Final ProductControls Performed on the Controls cells and on Supernatant fromControls Performed on the Controls cells and on Supernatant from
Production Cells of Purified BulkProduction Cells of Purified Bulk
Absence ofbacteria and fungi
Membrane filtration - According to EP(Transgene # C-0019)Bacterial and fungal sterility
TESTS ON SUPERNATANT FROM PRODUCTION CELLS
Absence ofextraneous agent
Inoculation of embryonated eggsAccording to EP
(Q-One Biotech # 37467)In vivo test for extraneous agent
Absence ofAvian Leukosis Virus
Inoculation of CEF and detection by ELISA(Transgene # C-0108)
Test forAvian Leukosis Virus
Absence of mycoplasmaCultivation assay and indicator DNA fluorochrome test - According to EP(Q-One Biotech # 38472&38471)Test for mycoplasma
Absence ofextraneous agents
Detection of cytopathogenic effect and hemadsorption on VERO, MRC5 and CEF cell cultures(Transgene # C-0086)
Tests in cell cultures for extraneous agents
Absence ofhemadsorbing viruses
Hemadsorption with Guinea red blood cells(Transgene # C-0007)
Test forhemadsorption viruses
Absence ofcytopathic effect
Microscopic observation(Transgene # C-0007)
Observation ofcontrol cells
CONTROL CELLS
Acceptance CriteriaMethodTests
Between 45 and 55 g/lRefractometry(Transgene # C-0101)Saccharose concentration
>30x104 pfu/ µgCalculationRatio between infectious titer and protein concentration
To be quantifiedModified Lowry method(Transgene # C-0096)Protein concentration
To be quantifiedELISA(Transgene # C-0093)Residual BSA content
Below or equal to 5 µg/mLMicrobiological assay(Q-One Biotech # 38344)Residual gentamycin content
Absence ofbacteria and fungi
Membrane filtrationAccording to EP
(Transgene # C-0019)Bacterial and fungal sterility
To be quantifiedPlaque-assay on BHK21 cells(Transgene # C-0078)Infectious titer
TESTS ON THE PURIFIED BULK
Absence of extraneous agentsInoculation of guinea-pigs
According to EP(Q-One Biotech # 37466)
In vivo tests for extraneous agents
Absence of extraneous agentsInoculation of mice, suckling-mice and Guinea pigs - According to EP(Q-One Biotech # 37465)In vivo tests for extraneous agents
Absence of extraneous agentsDetection of cytopathogenic effect and hemadorption on VERO, MRC5
cell cultures(Transgene # C-0086)
Tests in cell cultures for extraneous agents
Absence of mycoplasmaCultivation assay
According to EP(Q-One Biotech # 38472)
Test for mycoplasma
Absence ofbacteria and fungi
Membrane filtrationAccording to EP
(Transgene # C-0019)Bacterial and fungal sterility
To be quantifiedPlaque-assay on BHK21 cells(Transgene # C-0078)Infectious titer
TESTS ON CRUDE HARVEST
Acceptance CriteriaMethodTests
Between 250 and 350 Osm/kgDirect measurement(Transgene # A-0051)Osmolality
Between 7.4 and 8.2Direct measurement(Transgene # A-0005)pH
Absence ofabnormal toxicity
Inoculation of mice and guinea-pigsAccording to EP
(Q-One Biotech # 37003)Abnormal toxicity
<50 ng/doseCalculationResidual BSA content
<5 EU/mLLAL chromogenic assay(Q-One Biotech # 37195)Endotoxin content
Absence ofbacteria and fungi
Membrane filtrationAccording to EP
(Transgene # C-0019)Bacterial and fungal sterility
<1.0 log reduction in titerPlaque assay on BHK21 cells after exposure at 37°C during 7 daysAccelerated stability
To be quantifiedPlaque-assay on BHK21 cells(Transgene # C-0078)Infectious titer
Detection of expected deletion in excision region 3
PCR(Transgene # C0081)Identity of MVA strain
To be determined specificallyDepends on the nature of the transgeneExpression of foreign genes
Colorless to whitish limpid or slightly turbid liquidVisual observationVisual aspect
Tests on Final Bulk
Absence ofbacteria and fungi
Membrane filtrationAccording to EP
(Transgene # C-0019)Bacterial and fungal sterility
Test on Final Bulk
Acceptance CriteriaMethodTests
Automatic fillingin glass ampoules
FillingFilling
StorageStorage
L3 containment
Storage at -80°C, -20°C and -196°C
Packaging and shippingPackaging and shipping
Packaging according to IATA regulations for infectious material and shipping with dry ice.
Sain DMD
Gene Gene therapytherapy of DMD of DMD
Objective for gene-based therapy :≥ 20 % of the normal level
Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD) is an X-linkedgenetic disorder. Prevalence ranges from 1:3,000 to 1:3,500 boys.
Clinical symptoms become obvious at about 3 year of age.
Progressive muscle wasting leads to the loss of walking ability and wheelchair-dependence at about 10 years.
Respiratory insufficiency and cardiomyopathiesdramatically shorten life expectancy.
No curative treatment is available.
massive gene transfer
sustained expression
Genetic diseaseaffecting large tissue areas
UNFAVOURABLE CONTEXTUNFAVOURABLE CONTEXT
Option 1: PLASMID DNA AS VECTOROption 1: PLASMID DNA AS VECTOR
Safety / immuno-tolerance : no foreign proteins
Prolonged expression in skeletal muscle
Repeated administrations possible
Accommodate large genes (full-length dystrophin)
Large scale manufacturing available
cer
SV40 pA
dystrophin
ColE1
Kan r
16 kb
cer
SV40 pA
16S19SSV40 intron
ColE1
Kan r
cer
SV40 pA
Full-length
CMV
ColE1
Kan r
MyoDysMyoDys
Flow chart of plasmid DNA productionFlow chart of plasmid DNA productionFermentation
Cell harvest
Alcaline lysis
Clarification
Chromatography steps
Buffer exchange
Sterile filtration
Bulk product (API)
Plasmid DNA
Host strain selection
Fermentation optimization
Research cell bank
Research grade pilot
Quality and yield evaluation
GMP cell banking(MCB, WCB)
GMP manufacturing run
Quality control
Release
Filling
Untreated mdx5cv
Treated mdx5cv
dystrophin β-dystroglycan
pCMVpCMV--humanhuman dystrophindystrophin
GRMD dogs
Veterinary school Paris(St. Blot)
D+7, TA muscle
PositioningPositioning
whole limbs quality of life
respiratory muscles (diaphragm,…)breathing/expectoration, lifespan
1st step, quality of life
2nd and 3rd steps, quality of life + survival
forearm / hand muscles preserve control of wheelchair and keybords
Safety of the dystrophin plasmid vector :- low dose, local administration
Safety / efficacy of the delivery procedure :- higher dose,- intravascular administration to
a defined muscle territoryClinical efficacy :
- large muscle groups (limbs)- vital muscles (diaphragm, heart)
Clinical development strategy
Phase I clinical study of Phase I clinical study of dystrophindystrophin cDNAcDNA transfer using intramuscular transfer using intramuscular injection of plasmid DNA (TG5001) in injection of plasmid DNA (TG5001) in DuchenneDuchenne--type patientstype patients
Parameters : clinical, biology, biochemistryimmunology, inflammationdystrophin expressionhistology, muscle force
D0 D21
Recruitmentand follow-up
D90
biopsy
600 µg
D14
600 µg
# 1
# 2
# 3600 µg
200 µg biopsy
biopsy
biopsy
D-60
IntramuscularIntramuscular injection :injection : D 0D 0(Group 2 & 3)(Group 2 & 3)
IntramuscularIntramuscular injection :injection :
D 14 (Group 3)
DystrophinPositive controlmdx
Biopsy D+21 D-60
Patient R.R.
BlankPositive controlmdx
Biopsy D+21 D-60
Patient R.R.
Blank
Dystrophin mRNA
Phase I: Phase I: resultsresults
• 3 weeks after plasmid injection : - vector (plasmid) : 9/9 patients- dystrophin (histology + mRNA) : 6/9 patients
• Excellente safety profile (including immunology)
Romero et al. Hum. Gene Ther. 15: 1065-1076 (2004)
Trans gene
w heere rea d AFMn
NEXT STEP : SCALENEXT STEP : SCALE--UPUP
Intramuscular administrationlocal expression
NEXT STEP : SCALENEXT STEP : SCALE--UPUP
Intramuscular administrationlocal expression
Locoregional administrationhigher efficacy, topologyes
²²
Hagstrom et al. Mol. Ther. (2004)
Derived from the Bier’s Block procedure
Distribution of the Distribution of the plasmidplasmidpreparationpreparation in Macaque in Macaque rhesusrhesus limblimb
Blood vessel network is preserved
venous pressure
< 0.010.01 - 0. 1
0.1 - 11 - 55 - 10> 10
ng luc. / mg prot.
BF
Semitend
Rectusfemoris
tib. cran.
Semimemb
Abduct.
VASTUSLat. interm.
Medial.
gracilis
ERCEDC
Fl.unl.carp
Uln.lat.
ED.lat.
Fl.dig.superf.
Fl.dig.prof.
FRC
Pronatorteres
BF
EDLLon.peron. Poplit.
Fdmed.
Gastro.lat
Gastro.med
Canine dystrophinbiceps femoris (D+7)
luciferase
Sartorius
Biceps femoris
Contra-lateralEDC FCU IO BF
GRMD Normal
Impact on muscle force in Impact on muscle force in mdxmdx micemice#739 (EDL-Right Limb)
Time (sec)
0.4 0.6 0.8 1.0 1.2 1.4
Forc
e (g
ram
s)
0
5
10
15
20
25
30
35
#739 (EDL - Left Limb)
Time (sec)
0.4 0.6 0.8 1.0 1.2 1.4Fo
rce
(gra
ms)
0
5
10
15
20
25
30
35
Trial 1
Trial 3
Trial 5
Trial 5
Trial 3
Trial 1
Non treated Treated
Anti-dystrophin immune response- humoral response- no cellular infiltrates
Long term allogenic dystrophin expression- 1 year follow-up in mdx mice- 6 month follow-up in GRMD dogs
427kDa
+- + + +- - -+ -mAb PI I PI I
mouse # 1 mouse # 2
Immune rejection ?Immune rejection ?
- mouse dystrophin plasmids in mdx- canine dystrophin plasmids in GRMD
DeliveryDelivery procedureprocedure ((PathwayPathway IV) in IV) in humanshumans
Option 2: Exon Option 2: Exon skippingskipping
Type of mutations(from UMD-DMD data base – Institut Cochin)
Deletions (one or several exons) : >75%>75%
Duplications of part of the gene : >10%>10%
Nonsense mutations (Stop) : >5%>5%(mdx)
Micro-deletions/insersions : <5%<5%
Splice mutations : <5%<5% (GRMD)
2.5 million base pairs13,973 base pairs contain codingsequences / 79 exons
Exon Exon skippingskipping: an alternative: an alternative
Most mutations in the dystrophin gene create a frameshift or a stop in the mRNA and are associated with severe Duchenne muscular dystrophy.
Exon skipping that naturally occurs at low frequency sometimes eliminates the mutation and leads to the production of a rescued truncated protein.
The skipping of selected exons may remove the mutated exon on the dystrophinmessenger mRNA allowing production of a truncated potentially functional dystrophin.
Advantage: no risk of immune rejection of the newly producedtruncated protein
Exon chaining
A typical case of deletion: ∆51
Another typical case of deletion: ∆51-52
Rationale for Rationale for exonexon skippingskipping……
• Restore in-frame by elimination, during splicing, of the exons(s) responsiblefor the shift
Example : ∆48-50
AAV2 / modified-U7
INTRON.22…actcatcaaatatgcgtgttagtgtaaatgaacttctatttaattttgagGCTCTGCAAAGTTCTTTGAAAGAGCAACAAAATGGCTTCAACTATCTGAGTGACACTGTGAAGGAGATGGCCAAGAAAGCACCTTCAGAAATATGCCAGAAATATCTGTCAGAATTTGAAGAGATTGAGGGGCACTGGAAGAAACTTTCCTCCCAGTTGGTGGAAAGCTGCCAAAAGCTAGAAGAACATATGAATAAACTTCGAAAATTTCAGgtaagccgaggtttggcctttaaactatattttttcacatagcaattaat… INTRON.23
Branch point 22
SD23
Inserting antisense sequences in AAV-U7 carriers
Nonsense mutation > T (3185)
endogenous U7modified U7
+- +-15 days 1 month
+/+ mdx 0.5 1 32
dystrophin
Dys1-Ab Dys2-Ab
Missing domain encoded by exon 23
6 months
Dystrophin rescue (Western Blot)
Western blot of total protein extracted from injected mdx muscles probedwith the NCL-DYS1 monoclonal antibody.
Intra-arterial delivery of AAV vectors
1 ml/min(about 1012pp)
Transverse section of tibialis anterior muscle stained for AP activity after HPIA deliveryof AAV-1 muSEAP (murine secreted embryonic alkaline phosphatase) into hind-limb of normal mice.
Quasi-dystrophin expression following intra-arterial deliveryof AAV-U7 in mdx mice
NormalUntreated mdx15 days after injection AAV(U7-SD23/BP22)1 month after injection AAV(U7-SD23/BP22)2 months after injection AAV(U7-SD23/BP22)
4 months after injection AAV(U7-SD23/BP22)
One year after a single injection
Reduced damage in treated mdx muscle
untreated treated
Goyenvalle A, Vulin A, Fougerousse F, Leturcq F, Kaplan JC, Garcia L, Danos O.Science. (2004) 306:1796-1799
Multi-skipping in GRMD (in vivo studies)
1 month after intramuscular injection of AAV(U7 C6ESE2) & AAV(U7C8ESE1).
SummarySummary
• Efficient and stable exon-skipping on the dystrophin pre-mRNA can be obtained usingAAV vectors harboring modified U7 snRNAs (at least one year).
• AAV vectors are compatible with systemic delivery.
• The rescued quasi-dytrophin is fully functional (at least in mdx and probably in appropriate DMD genotypes).
• The final product (quasi-dystrophin) is not rejected by the immune system.
Issues:
-Allele specific strategy (« à la carte »), not every patient is eligible, safetyissues inherent to gene based strategies...
-Avoid rejection of the vector…
RegulatoryRegulatory processprocess
SPONSORFile
AFSSAPS
Evaluation:- gene therapy group- viral security group
Autorisation
Sponsor
C.G.G.(Ministère de la recherche)
C.G.B.(Ministère de l’Agriculture
Ministère de l’environnement)
CCPPRB(P.I.)
Clinical site
Control of dissemination
OGM classificationAgreement
OGM > 60days
Oral Oral rabiesrabies vaccinevaccine
RaboralTM
Rabies glycoprotein gene
Modified vaccinia virus
baits
Oral Oral rabiesrabies vaccine vaccine
RaboralTM
Rabies glycoprotein gene
Modified vaccinia virus
Oral Oral rabiesrabies vaccine vaccine
RaboralTM
Rabies glycoprotein gene
Modified vaccinia virus
Eradication of Eradication of rabiesrabies diseasedisease in the in the wildwild lifelife
RaboralTM
1989 1997
2001: in France
Major Success Stories andMajor Success Stories and SetbacksSetbacks
Retrovirus-based treatment of infants suffering from the X-chromosome-linked severe combined immunodeficiency disease (SCID) (bubble children). Following this treatment, these children have been able to live in the open air.
Setbacks:Three of the SCID-XI-treated patients developed a leukemia-like
condition.
One fatal case of adenovirus-based treatment of a non-life-threatening disease, Ornithine Transcarbamylase deficiency.
« Nous devons nous rappeler que l’évolution de la pratique médicale est dynamique. A chaque fois qu’un effet secondaire imprévu se produit, une évaluation est à nouveau entreprise, et de ce processus émergent de nouvelles idées scientifiques qui, au bout du compte, génèrent de nouvelles médecines utiles ».
« We must remember that the evolution of medicalpractice is dynamic. As each new, unexpected adverse event arises, evaluation is carried out again, and in the process, new scientific ideas unfold that ultimatelyyield medically useful products ».
Philip Noguchi (FDA, Food and Drug Administration)
Gene Gene therapytherapy :: reasonsreasons for for believingbelieving
Higher maturity of vector technology
Not unrisky, but safer
Indications of efficacy in some applications
One approved product (GenedicineTM), severalPhase III trials
Gene Gene therapytherapy :: reasonsreasons for for believingbelieving
Obstination leads to success. Therefore, the more failures, the higher chances are that it’s going to work.