ppt for dna vaccine production

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.