Gene therapy :

from dream to (tough) reality

Serge Braun, AFM

GENE THERAPY : Transfer of genes into cells to prevent or

treat diseases

Local or systemic administration Hereditary or acquired diseases

An 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/Lentivirus Alphavirus Measles Herpes simplex Adenovirus AAV Poxvirus Salmonella typhimurium Listeria monocytogenes Plasmid (naked DNA) Electroporation Sonoporation Gene gun Lipoplexes Peptidoplexes Solvoplexes RNA transfer

Vectors

Retrovirus

Adenovirus

Poxvirus

+ -

Integration Safety chronic diseases

+ Level of -

expression Readministration ? non chronic

+ -

Efficacy Transient expression

+ No immune response + Repeated administr. - Low efficacy

Long term

Non-viral

Vaccines

Targeting of different organs by viral vectors

Lundstrom (2003) Trends in Biotechnology, 21: 117.

Construction of Vectors

Adenovirus type 5 genome

E1A E1B
ITR

L1
MLP

L2

L3

L4 E3

L5

3 5

pIX

ITR

IVa2

5 E2A (DBP) 3 E4

Early

E2B (pTP, POL)

E1A, E1B : E2A, E2B : E2A : E4 : transcription, transactivation, immortalization, transformation viral DNA synthesis transactivation of MLP regulation of : DNA synthesis, early / late transcription, mRNA stability, splicing, apoptosis immunomodulation : downregulation of MHC capsid protein, transactivation viral structural proteins, viral assembly

Late

E3 : pIX: MLP (L1 - L5) :

Adenovirus as gene transfer vector

Left ITR E1

Ad 5 genome

E3

Right ITR

Promoter

Intron

Therapeutic Gene

pA

Expression cassette

Production in a complementing cell line

Left ITR E1

Ad 5 genome

E3

Right ITR

Promoter

Intron

Therapeutic Gene

pA

Complementing cell line

E1/E3

Production in a complementing cell line

Left ITR E1

Ad 5 genome

E3

Right ITR

Promoter

Intron

Therapeutic Gene

pA

Complementing cell line

E1/E3

Production in a complementing cell line

Left ITR E1

Ad 5 genome

E3

Right ITR

Promoter

Intron

Therapeutic Gene

pA

Complementing cell line

E1/E3

Production in a complementing cell line

Left ITR E1

Ad 5 genome

E3

Right ITR

Promoter

Intron

Therapeutic Gene

pA

Complementing cell line

E1/E3

AAV

Regulatory aspects:
GMPs: products of biological origin Containment and GMOs: protect personnel and avoid dissemination: L3 Conciliate GMPs and containment

Large scale production of recombinant virus

Bioreactor, suspended cells

Clarification (Filtration)

Chemical elimination of enveloped viruses

Purification

Ion exchange chromatography

Bioprocess apparatus

Purification

Buffers in disposable pallets tanks

Molecular sifting

HPLC analysis
Raw After chromatography

Formulated product

Controls throughout the process

Raw material Cell, virus banks Identity, efficacy, stability

FERMENTATION

Bulk

Purity Absence of viral contaminants

PURIFICATION

Purified bulk
FORMULATION AND REPARTITION

Purity Dosage of residuals

Final product

Identity, efficacy, stability

Quality control of the Final Product

Controls Performed on the Controls cells and on Supernatant from Production Cells of Purified Bulk
Tests Method CONTROL CELLS Observation of control cells Test for hemadsorption viruses Tests in cell cultures for extraneous agents Microscopic observation (Transgene # C-0007) Hemadsorption with Guinea red blood cells (Transgene # C-0007) Detection of cytopathogenic effect and hemadsorption on VERO, MRC5 and CEF cell cultures (Transgene # C-0086) Cultivation assay and indicator DNA fluorochrome test - According to EP (Q-One Biotech # 38472&38471) Inoculation of CEF and detection by ELISA (Transgene # C-0108) Inoculation of embryonated eggs According to EP (Q-One Biotech # 37467) TESTS ON SUPERNATANT FROM PRODUCTION CELLS Bacterial and fungal sterility Membrane filtration - According to EP (Transgene # C-0019) Absence of bacteria and fungi Absence of cytopathic effect Absence of hemadsorbing viruses Absence of extraneous agents Acceptance Criteria

Test for mycoplasma

Absence of mycoplasma

Test for Avian Leukosis Virus

Absence of Avian Leukosis Virus Absence of extraneous agent

In vivo test for extraneous agent

Tests

Method TESTS ON CRUDE HARVEST

Acceptance Criteria

Infectious titer

Plaque-assay on BHK21 cells (Transgene # C-0078) Membrane filtration According to EP (Transgene # C-0019) Cultivation assay According to EP (Q-One Biotech # 38472) Detection of cytopathogenic effect and hemadorption on VERO, MRC5 cell cultures (Transgene # C-0086) Inoculation of mice, suckling-mice and Guinea pigs - According to EP (Q-One Biotech # 37465)

To be quantified

Bacterial and fungal sterility

Absence of bacteria and fungi

Test for mycoplasma

Absence of mycoplasma

Tests in cell cultures for extraneous agents

Absence of extraneous agents

In vivo tests for extraneous agents

Absence of extraneous agents

In vivo tests for extraneous agents

Inoculation of guinea-pigs According to EP (Q-One Biotech # 37466) TESTS ON THE PURIFIED BULK

Absence of extraneous agents

Infectious titer

Plaque-assay on BHK21 cells (Transgene # C-0078) Membrane filtration According to EP (Transgene # C-0019) Microbiological assay (Q-One Biotech # 38344) ELISA (Transgene # C-0093) Modified Lowry method (Transgene # C-0096) Calculation Refractometry (Transgene # C-0101)

To be quantified

Bacterial and fungal sterility

Absence of bacteria and fungi

Residual gentamycin content

Below or equal to 5 g/mL

Residual BSA content

To be quantified

Protein concentration Ratio between infectious titer and protein concentration Saccharose concentration

To be quantified

>30x104 pfu/ g

Between 45 and 55 g/l

Tests

Method Test on Final Bulk Membrane filtration According to EP (Transgene # C-0019) Tests on Final Bulk

Acceptance Criteria

Bacterial and fungal sterility

Absence of bacteria and fungi

Visual aspect Expression of foreign genes Identity of MVA strain

Visual observation Depends on the nature of the transgene PCR (Transgene # C0081) Plaque-assay on BHK21 cells (Transgene # C-0078) Plaque assay on BHK21 cells after exposure at 37C during 7 days Membrane filtration According to EP (Transgene # C-0019) LAL chromogenic assay (Q-One Biotech # 37195) Calculation Inoculation of mice and guinea-pigs According to EP (Q-One Biotech # 37003) Direct measurement (Transgene # A-0005) Direct measurement (Transgene # A-0051)

Colorless to whitish limpid or slightly turbid liquid To be determined specifically Detection of expected deletion in excision region 3 To be quantified

Infectious titer

Accelerated stability

<1.0 log reduction in titer

Bacterial and fungal sterility

Absence of bacteria and fungi

Endotoxin content Residual BSA content

<5 EU/mL <50 ng/dose Absence of abnormal toxicity

Abnormal toxicity

pH

Between 7.4 and 8.2

Osmolality

Between 250 and 350 Osm/kg

Filling
Automatic filling in glass ampoules

Storage

L3 containment

Storage at -80C, -20C and -196C

Packaging and shipping

Packaging according to IATA regulations for infectious material and shipping with dry ice.

Gene therapy of DMD

Sain

DMD

Objective for gene-based therapy : 20 % of the normal level

Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is an X-linked genetic 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 cardiomyopathies dramatically shorten life expectancy. No curative treatment is available.

UNFAVOURABLE CONTEXT
Genetic disease affecting large tissue areas massive gene transfer sustained expression

Option 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
CMV
ColE1 cer Kan
r

SV40 pA

MyoDys
16 kb Full-length dystrophin

16S19S SV40 intron

Flow chart of plasmid DNA production

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 Fermentation Cell harvest Alcaline lysis Clarification Chromatography steps Buffer exchange Bulk product (API) Sterile filtration Filling

pCMV-human dystrophin

GRMD dogs
Veterinary school Paris (St. Blot)

dystrophin

-dystroglycan

Untreated mdx5cv
D+7, TA muscle

Treated mdx5cv

Positioning
1st step, quality of life
forearm / hand muscles preserve control of wheelchair and keybords

2nd and 3rd steps, quality of life + survival

whole limbs quality of life breathing/expectoration, lifespan

respiratory muscles (diaphragm,)

Clinical development strategy

Safety of the dystrophin plasmid vector : - low dose, local administration Safety / efficacy of the delivery procedure : - higher dose, - intravascular administration to a defined muscle territory Clinical efficacy : - large muscle groups (limbs) - vital muscles (diaphragm, heart)

Phase I clinical study of dystrophin cDNA transfer using intramuscular injection of plasmid DNA (TG5001) in Duchenne-type patients
D-60
Recruitment and follow-up

D0 #1 #2
200 g

D14

D21 biopsy biopsy biopsy

D90

biopsy

600 g

600 g

600 g

#3

Parameters :

clinical, biology, biochemistry immunology, inflammation dystrophin expression histology, muscle force

Intramuscular injection : D 0
(Group 2 & 3)

Intramuscular injection : D 14
(Group 3)

Phase I: results
Dystrophin mRNA

Dystrophin

Positive control mdx

Biopsy D+21

D-60

Blank

Patient R.R.

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)

Tr an s g e ne a nd A FM wer e h e re

NEXT STEP : SCALE-UP

Intramuscular administration local expression

NEXT STEP : SCALE-UP

Intramuscular administration local expression Locoregional administration higher efficacy, topologyes

 Derived from the Biers Block procedure

Hagstrom et al. Mol. Ther. (2004)

Distribution of the plasmid preparation in Macaque rhesus limb

Blood vessel network is preserved

venous pressure

luciferase

Rectus femoris

VASTUS interm. Lat. Medial. Sartorius BF Abduct. Semi Semi memb

tend

gracilis

Canine dystrophin
biceps femoris (D+7) ng luc. / mg prot. < 0.01 0.01 - 0. 1 0.1 - 1 1-5 5 - 10 > 10 ERC
Pronator teres

tib. cran. EDC

EDL ED.lat.
Uln. lat. BF
Gastro. lat Lon.peron.

Poplit.
Fdmed.

FRC Fl.dig.prof.

Gastro. med

Fl.unl.carp Fl.dig.superf.

Biceps femoris

EDC

FCU IO GRMD

BF Normal

Contra-lateral

Impact on muscle force in mdx mice

#739 (EDL-Right Limb) 35 30 25 35 30 25 #739 (EDL - Left Limb)

Force (grams)

Force (grams)

20 15 10
Trial 3 Trial 1

20 15 10

Trial 1

Trial 3

Trial 5

5
Trial 5

5 0 0.4 0.6 0.8 1.0 1.2 1.4 0.4 0.6 0.8 1.0 1.2 1.4

Time (sec)

Time (sec)

Non treated

Treated

Immune rejection ?
- mouse dystrophin plasmids in mdx - canine dystrophin plasmids in GRMD
427 kDa

mouse # 1 mouse # 2 mAb PI I PI I

+ - - + - + - + - +

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

Delivery procedure (Pathway IV) in humans

Option 2: Exon skipping

2.5 million base pairs 13,973 base pairs contain coding sequences / 79 exons

Type of mutations
(from UMD-DMD data base Institut Cochin) Deletions (one or several exons) : >75% Duplications of part of the gene : >10% Nonsense mutations (Stop) : >5% (mdx) Micro-deletions/insersions : <5% Splice mutations : <5% (GRMD)

Exon skipping: 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 dystrophin messenger mRNA allowing production of a truncated potentially functional dystrophin.

Advantage: no risk of immune rejection of the newly produced truncated protein

Exon chaining

A typical case of deletion: 51

Another typical case of deletion: 51-52

Rationale for exon skipping

Restore in-frame by elimination, during splicing, of the exons(s) responsible for the shift

Example : 48-50

Inserting antisense sequences in AAV-U7 carriers

Nonsense mutation > T (3185)

Branch point 22

INTRON.22actcatcaaatatgcgtgttagtgtaaatgaacttctatttaattttgagGCTCTGCAAAGTTCTTTGAAAGAGCAACAAAATGGCTTCAACTATCT GAGTGACACTGTGAAGGAGATGGCCAAGAAAGCACCTTCAGAAATATGCCAGAAATATCTGTCAGAATTTGAAGAGATTGAGGGGCAC TGGAAGAAACTTTCCTCCCAGTTGGTGGAAAGCTGCCAAAAGCTAGAAGAACATATGAATAAACTTCGAAAATTTCAGgtaagccgaggtttg gcctttaaactatattttttcacatagcaattaat INTRON.23 SD23

AAV2 / modified-U7

15 days

1 month

modified U7 endogenous U7

Dystrophin rescue (Western Blot)

Western blot of total protein extracted from injected mdx muscles probed with the NCL-DYS1 monoclonal antibody. +/+
dystrophin

mdx

0.5

6 months

Missing domain encoded by exon 23

Dys1-Ab

Dys2-Ab

Intra-arterial delivery of AAV vectors

1 ml/min (about 1012pp)
Transverse section of tibialis anterior muscle stained for AP activity after HPIA delivery of AAV-1 muSEAP (murine secreted embryonic alkaline phosphatase) into hind-limb of normal mice.

Quasi-dystrophin expression following intra-arterial delivery of AAV-U7 in mdx mice

Normal after injection AAV(U7-SD23/BP22) Untreated mdx injection AAV(U7-SD23/BP22) 15 monthafter injection AAV(U7-SD23/BP22) 12 days months after

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(U7 C8ESE1).

Summary
Efficient and stable exon-skipping on the dystrophin pre-mRNA can be obtained using AAV 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 ( la carte ), not every patient is eligible, safety issues inherent to gene based strategies... -Avoid rejection of the vector

Regulatory process
SPONSOR File C.G.G.
(Ministre de la recherche) Agreement OGM > 60days OGM classification

AFSSAPS

C.G.B.
(Ministre de lAgriculture Ministre de lenvironnement) Control of dissemination

Clinical site

Evaluation: - gene therapy group - viral security group Autorisation Sponsor

CCPPRB (P.I.)

Oral rabies vaccine

RaboralTM

Rabies glycoprotein gene

Modified vaccinia virus

Oral rabies vaccine

RaboralTM

Rabies glycoprotein gene

baits
Modified vaccinia virus

Oral rabies vaccine

RaboralTM

Rabies glycoprotein gene

Modified vaccinia virus

Eradication of rabies disease in the wild life RaboralTM

1989 1997

2001: in France

Major Success Stories and Setbacks

Retrovirus-based treatment of infants suffering from the X-chromosomelinked 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.

 We must remember that the evolution of medical practice is dynamic. As each new, unexpected adverse event arises, evaluation is carried out again, and in the process, new scientific ideas unfold that ultimately yield medically useful products .
Philip Noguchi (FDA, Food and Drug Administration)

Nous devons nous rappeler que lvolution de la pratique mdicale est dynamique. A chaque fois quun effet secondaire imprvu se produit, une valuation est nouveau entreprise, et de ce processus mergent de nouvelles ides scientifiques qui, au bout du compte, gnrent de nouvelles mdecines utiles .

Gene therapy : reasons for believing

Higher maturity of vector technology Not unrisky, but safer Indications of efficacy in some applications One approved product (GenedicineTM), several Phase III trials

Gene therapy : reasons for believing

Obstination leads to success. Therefore, the more failures, the higher chances are that its going to work.