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Chiron CorporationChiron CorporationCenter for Gene TherapyCenter for Gene Therapy

Emeryville and San Diego, CaliforniaEmeryville and San Diego, California

Deborah Hurst, MDDeborah Hurst, MDDirector, Clinical DevelopmentDirector, Clinical Development

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Overview : Risk of Germ Line Transduction Overview : Risk of Germ Line Transduction After Direct Injection of Retroviral Vectors After Direct Injection of Retroviral Vectors

History of retroviral vectors in the clinic History of retroviral vectors in the clinic

Rationale for current use of retroviral vector by Rationale for current use of retroviral vector by intravenous route for hemophilia A (FVIII deficiency)intravenous route for hemophilia A (FVIII deficiency)

Preclinical data on germ line transduction Preclinical data on germ line transduction supporting use of FVIII vector in mansupporting use of FVIII vector in man

Clinical data from ongoing FVIII trialClinical data from ongoing FVIII trial

Proposal for follow-up Proposal for follow-up

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Chiron’s Use of Retroviral Vectors in Clinical Chiron’s Use of Retroviral Vectors in Clinical DevelopmentDevelopment

1993-99: 1993-99: Ex-vivoEx-vivo transduction (cancer) transduction (cancer) – 3 trials / 33 subjects3 trials / 33 subjects

1994-98: Intratumor/I.M. 1994-98: Intratumor/I.M. injections (cancer, injections (cancer, HIV) HIV) – 7 trials / 250 subjects7 trials / 250 subjects

1999- ongoing: I.V. infusion (Hemophilia A)1999- ongoing: I.V. infusion (Hemophilia A)– 1 trial / 13 subjects1 trial / 13 subjects

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Past Testing for Germ Line TransmissionPast Testing for Germ Line Transmission During Clinical Use of Retroviral Vectors During Clinical Use of Retroviral Vectors

104 subjects tested for vector in semen104 subjects tested for vector in semen

Maximum I.M. dose approximately 100-fold less Maximum I.M. dose approximately 100-fold less than the current I.V. dose than the current I.V. dose

Specimens tested by PCR assay with single copy Specimens tested by PCR assay with single copy sensitivity sensitivity

All samples negativeAll samples negative

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Choice of Retroviral Vector and Choice of Retroviral Vector and I.V. Delivery for Factor VIII Gene TransferI.V. Delivery for Factor VIII Gene Transfer

Retroviral vectorRetroviral vector– Stable integration, potential long-term expressionStable integration, potential long-term expression– Excellent safety record Excellent safety record – Transduction resulted in therapeutic FVIII levels in preclinical Transduction resulted in therapeutic FVIII levels in preclinical

models models

Intravenous delivery Intravenous delivery – Non-invasive (hand vein)Non-invasive (hand vein)

patients prone to bleeding, liver dysfunctionpatients prone to bleeding, liver dysfunction– Targets cells in direct contact with bloodTargets cells in direct contact with blood

secreted FVIII must bind to vWF and circulatesecreted FVIII must bind to vWF and circulate multiple tissues can produce FVIII multiple tissues can produce FVIII

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Derived from Moloney Murine Leukemia VirusDerived from Moloney Murine Leukemia Virus

Replication deficientReplication deficient

Carries gene for human Factor VIII protein Carries gene for human Factor VIII protein

Human complement-resistantHuman complement-resistant

Manufactured at high titer (10Manufactured at high titer (1088-10-1099 TU/ml) TU/ml)

Retroviral Vector Expressing Human Retroviral Vector Expressing Human Factor FVIII [hFVIII(V)]Factor FVIII [hFVIII(V)]

5'LTR B-domain deleted hFVIII 3'LTR

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Preclinical Biolocalization Studies Using Preclinical Biolocalization Studies Using PCR to Detect Vector Sequences PCR to Detect Vector Sequences

122 rabbits, 4 hemophilic dogs received I.V. vector 122 rabbits, 4 hemophilic dogs received I.V. vector

Tissue samples obtained at different timepoints after infusionTissue samples obtained at different timepoints after infusion

PCR assay with single copy sensitivity PCR assay with single copy sensitivity

Multiple replicates tested (1Multiple replicates tested (1g DNA/replicate)g DNA/replicate)~ 150,000 diploid genome equivalents/replicate ~ 150,000 diploid genome equivalents/replicate

Statistical assumptionsStatistical assumptions– Reactions independent of each otherReactions independent of each other

– Sampling representative of the vector distribution in the specimenSampling representative of the vector distribution in the specimen

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Summary of Biolocalization StudiesSummary of Biolocalization Studies in Rabbits and Hemophilic Dogs in Rabbits and Hemophilic Dogs

Localization of vector sequences after IV injection:Localization of vector sequences after IV injection:

- liver / spleen- liver / spleen high signal persists to 2 yearshigh signal persists to 2 years

- bone marrow / - bone marrow / PBMCPBMC high signal declines over timehigh signal declines over time

- lung / thymus / kidney - lung / thymus / kidney intermittent, threshold signalintermittent, threshold signal lymph node / testislymph node / testis

- brain- brain negativenegative

- semen- semen negative in dogs (weeks 22 and 98) negative in dogs (weeks 22 and 98) and rabbits (through 21 weeks)and rabbits (through 21 weeks)

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Probability of a Transduced Cell in Rabbit Probability of a Transduced Cell in Rabbit Testis Is Very Low and Decreases Over TimeTestis Is Very Low and Decreases Over Time

Time after vector infusion (days)

Pro

bab

ility

of

a t

ran

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l0.000020

0.000015

0.000010

0.000005

0.00000015 7274 90

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Semen Analysis Study DesignSemen Analysis Study Design

Adult male rabbits (N=13) treated with hFVIII(V)Adult male rabbits (N=13) treated with hFVIII(V) Semen samples collected weekly for PCR testingSemen samples collected weekly for PCR testing Duration: 21 weeks to span multiple cycles of Duration: 21 weeks to span multiple cycles of

spermatogenesisspermatogenesis Clinically-relevant doses given I.V. to ensureClinically-relevant doses given I.V. to ensure

– therapeutic hFVIII levels in bloodtherapeutic hFVIII levels in blood

– positive PCR signal in testispositive PCR signal in testis

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Results of Rabbit Semen Study Results of Rabbit Semen Study

No positive semen samples No positive semen samples – For each sample: 20 replicates, 2 primer setsFor each sample: 20 replicates, 2 primer sets

Incidence of sporadic, unconfirmed signalsIncidence of sporadic, unconfirmed signalsuntreated controls = 0.00125 (1/800)untreated controls = 0.00125 (1/800)

treated animals = 0.00070 (3/4281)treated animals = 0.00070 (3/4281)

Thus, false positive PCR rate = 0.078%Thus, false positive PCR rate = 0.078%– lower than published rates from other labslower than published rates from other labs

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PCR Results in Rabbit PCR Results in Rabbit Testes and SemenTestes and Semen

TestesTestes After one cycle of spermatogenesis (90 days), risk of After one cycle of spermatogenesis (90 days), risk of

any single rabbit testis cell containing the vector any single rabbit testis cell containing the vector genome was 1 in 937,000 (1 in 709,000, 99% genome was 1 in 937,000 (1 in 709,000, 99%

confidence limit).confidence limit).

SemenSemen All samples negative throughout weekly testing over All samples negative throughout weekly testing over

multiple cycles of spermatogenesis multiple cycles of spermatogenesis

Conclusion:Conclusion: Positive PCR for retroviral vector in gonadal tissue Positive PCR for retroviral vector in gonadal tissue was not associated with transduced sperm cells in was not associated with transduced sperm cells in semen.semen.

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Phase I Multicenter Study of FVIII Retroviral Phase I Multicenter Study of FVIII Retroviral Vector in Subjects With Severe Hemophilia A Vector in Subjects With Severe Hemophilia A

Endpoints: Safety, circulating FVIII levels Endpoints: Safety, circulating FVIII levels

Study design:Study design:– Single treatment - IV infusions, days 1-3Single treatment - IV infusions, days 1-3– Dose escalation (2.7 x 10Dose escalation (2.7 x 1077 to 8.8 x 10 to 8.8 x 1088 TU/kg) TU/kg)– Subjects: Adult men with severe Hemophilia ASubjects: Adult men with severe Hemophilia A– Informed consent: barrier contraception; risk of IGTInformed consent: barrier contraception; risk of IGT– Semen test points: pre-infusion, weeks 2, 6, 9, 11, 17, 29, 53 Semen test points: pre-infusion, weeks 2, 6, 9, 11, 17, 29, 53

(plus additional samples in case of positive result) (plus additional samples in case of positive result) – Duration: one yearDuration: one year

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Current Study StatusCurrent Study Status

12 subjects on study for at least 3 months 12 subjects on study for at least 3 months

4 dose levels administered, 3 subjects at each4 dose levels administered, 3 subjects at each

Infusions well-toleratedInfusions well-tolerated

All subjects alive, in their usual states of healthAll subjects alive, in their usual states of health

Semen PCR results available from Semen PCR results available from – 11 subjects through week 17 11 subjects through week 17 – 8 subjects through week 298 subjects through week 29– 3 subjects through week 533 subjects through week 53

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Clinical PCR Assay for Vector in Semen Clinical PCR Assay for Vector in Semen

10 replicates tested per sample, 1 10 replicates tested per sample, 1 g DNA/replicateg DNA/replicate

Single copy sensitivitySingle copy sensitivity

Assay procedures designed to minimize common Assay procedures designed to minimize common sources of contamination leading to false positivessources of contamination leading to false positives

Collections scheduled to maximize chance of Collections scheduled to maximize chance of detecting transduced sperm detecting transduced sperm

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Spermatogenesis Model Supporting theSpermatogenesis Model Supporting the Semen Testing Protocol Semen Testing Protocol

Germ cells accessible to blood-borne RVV must be Germ cells accessible to blood-borne RVV must be dividing and on blood side of Sertoli-cell barrierdividing and on blood side of Sertoli-cell barrier– Differentiating spermatogonia (many/rapidly dividing), Differentiating spermatogonia (many/rapidly dividing),

dividing stem cells (few)dividing stem cells (few)– NOT spermatocytes, spermatids, or spermatozoaNOT spermatocytes, spermatids, or spermatozoa

Timing of transduced sperm in semen is predictable Timing of transduced sperm in semen is predictable – Interval based upon time required for maturation from Interval based upon time required for maturation from

transduced precurser transduced precurser – 49-92 days after vector infusion (study weeks 8-14)49-92 days after vector infusion (study weeks 8-14)

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Time Points for Semen Collection Time Points for Semen Collection During First Cycle of Spermatogenesis During First Cycle of Spermatogenesis

Vector Vector infusionsinfusions

Wk 18Wk 18

First possible First possible appearance in semen appearance in semen of transduced spermof transduced sperm

Last sperm in semen Last sperm in semen from first cycle of from first cycle of spermatogenesisspermatogenesis

00

Peak appearancePeak appearance

Per protocol, additional samples are obtained Per protocol, additional samples are obtained following any positive test result until three following any positive test result until three consecutive samples test negative.consecutive samples test negative.

= Scheduled semen = Scheduled semen collectioncollection

Wk 0Wk 0 Wk 2Wk 2 Wk 6Wk 6 Wk 9Wk 9 Wk 11Wk 11 Wk 17Wk 17

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Human Semen PCR Results to DateHuman Semen PCR Results to Date

63 semen samples tested from 11 subjects63 semen samples tested from 11 subjects

Results: Results: 61 negative61 negative– 1 positive (1/10 replicates tested)1 positive (1/10 replicates tested)– 1 indeterminate 1 indeterminate (contamination suspected)(contamination suspected)

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Semen Test Results Semen Test Results Subject #01001 / F8V101Subject #01001 / F8V101

Vector Vector infusioninfusion

Wk 18Wk 18

First possible First possible appearance in appearance in semen of transduced semen of transduced spermsperm

Last sperm in Last sperm in semen from first semen from first cycle of cycle of spermatogenesisspermatogenesis

00Peak Peak

appearanceappearance

Wk 0Wk 0 Wk 2Wk 2 Wk 6Wk 6 Wk 9Wk 9 Wk 11Wk 11 Wk 12Wk 12 Wk 14Wk 14 Wk 17Wk 17

Positive (1/10)Positive (1/10)NegativeNegative

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Possible Sources of Positive PCRPossible Sources of Positive PCRTest Result in Subject #01001Test Result in Subject #01001

Transduced somatic cell Transduced somatic cell – Semen contains PMNs, macrophages, lymphocytes, epithelial cellsSemen contains PMNs, macrophages, lymphocytes, epithelial cells– Subject’s PBMC are PCR-positive for vectorSubject’s PBMC are PCR-positive for vector

Test contaminant Test contaminant

Sperm produced from transduced spermatogoniaSperm produced from transduced spermatogonia– Low frequency - 1 positive out of 10 replicatesLow frequency - 1 positive out of 10 replicates– Detected during first cycle of spermatogenesis Detected during first cycle of spermatogenesis – Repeat samples negativeRepeat samples negative

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Pooled data - all timepoints 1 / 1901 / 190

One in One in 57,000,00057,000,000

No. Positive/No. Positive/No. Replicates No. Replicates

TestedTested

Probability of a Probability of a Sperm Cell Sperm Cell

Being Being TransducedTransduced

Pooled high risk data - weeks 9 & 11 1 / 701 / 70

One in One in 21,000,00021,000,000

One inOne in8,600,0008,600,000

99% 99% Confidence Confidence

BoundBound

One in One in 3,100,000 3,100,000

Frequency Analysis of Human Semen Test Frequency Analysis of Human Semen Test Results From Dose Level 4*Results From Dose Level 4*

**Highest dose completed to dateHighest dose completed to date = 4.4 x 10= 4.4 x 1088 TU/kg TU/kg

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Results of Human Semen PCR Analysis Results of Human Semen PCR Analysis

At a 99% confidence level, the worst case frequency of a transduced At a 99% confidence level, the worst case frequency of a transduced sperm is:sperm is:

1 in 3,100,000 - 8,600,000 cells1 in 3,100,000 - 8,600,000 cells

or 0.0000003 to 0.0000001or 0.0000003 to 0.0000001

No positive samples have been detected after the first cycle of No positive samples have been detected after the first cycle of spermatogenesisspermatogenesis

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ConclusionsConclusions

Current human data in semen is consistent with preclinical Current human data in semen is consistent with preclinical data: the probability of a germline cell being transduced is data: the probability of a germline cell being transduced is very low.very low.

The probability of inadvertent germline transmission of this The probability of inadvertent germline transmission of this retroviral vector genome at the current dose is remote.retroviral vector genome at the current dose is remote.

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Proposed Follow-Up for F8V101 Subjects With Proposed Follow-Up for F8V101 Subjects With PCR-Positive Semen Testing PCR-Positive Semen Testing

Repeat tests negative x 3 over three months and Repeat tests negative x 3 over three months and negative at study end (1 year)negative at study end (1 year)– No additional semen collection required No additional semen collection required

Repeat tests positive, or sporadically positive, after first Repeat tests positive, or sporadically positive, after first 3 months and to study end 3 months and to study end – Perform WBC/sperm cell separation, if feasiblePerform WBC/sperm cell separation, if feasible– Estimate risk of germ line transmission based on signal Estimate risk of germ line transmission based on signal

frequencyfrequency– Provide genetic counseling re. possible outcomes of Provide genetic counseling re. possible outcomes of

conception and reproductive alternatives conception and reproductive alternatives

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ChironChiron

Vector R & DVector R & DNicholas DePolo,Nicholas DePolo, Carlos Ibanez, Carlos Ibanez, Douglas Jolly, Sybille SauterDouglas Jolly, Sybille Sauter

Vector ProductionVector ProductionK. Jon Kowal, James Marich, Holger K. Jon Kowal, James Marich, Holger Roehl, Paula Stemler Roehl, Paula Stemler

PreclinicalPreclinicalMartin Giedlin, Martha Leibbrandt, Martin Giedlin, Martha Leibbrandt, Joanne Rose LayshockJoanne Rose Layshock

ClinicalClinicalVeronica Cole, Deborah Hurst, Veronica Cole, Deborah Hurst,

Biao Lu Biao Lu

DevelopmentDevelopment Donald Gay, Biff Owen Donald Gay, Biff Owen

Clinical InvestigatorsClinical Investigators Jeanne Lusher MDJeanne Lusher MD

Wayne State U. Medical Center, Detroit Wayne State U. Medical Center, Detroit

Jerry S. Powell MDJerry S. Powell MDU. California Davis Medical Center, SacramentoU. California Davis Medical Center, Sacramento

Margaret Ragni MD, MPHMargaret Ragni MD, MPHU. Pittsburgh Medical Center, PittsburghU. Pittsburgh Medical Center, Pittsburgh

Gilbert White MDGilbert White MDU. of North Carolina Medical Center, Chapel HillU. of North Carolina Medical Center, Chapel Hill

Contributors - FVIII StudyContributors - FVIII Study

ConsultantConsultant Kim Boekelheide, MD, PhDKim Boekelheide, MD, PhD Brown U., ProvidenceBrown U., Providence