# Presentation: 509365

## Chiron CorporationCenter for Gene Therapy

- Emeryville and San Diego, California
- Deborah Hurst, MD
- Director, Clinical Development

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

- History of retroviral vectors in the clinic
- Rationale for current use of retroviral vector by intravenous route for hemophilia A (FVIII deficiency)
- Preclinical data on germ line transduction supporting use of FVIII vector in man
- Clinical data from ongoing FVIII trial
- Proposal for follow-up

## Chiron’s Use of Retroviral Vectors in Clinical Development

- 1993-99:  *Ex-vivo* transduction (cancer)
- 3 trials / 33 subjects
- 1994-98:  Intratumor/I.M. injections (cancer, HIV)
- 7 trials / 250 subjects
- 1999- ongoing:   I.V. infusion (Hemophilia A)
- 1 trial / 13 subjects

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

- 104 subjects tested for vector in semen
- Maximum I.M. dose approximately 100-fold less than the current I.V. dose
- Specimens tested by PCR assay with single copy sensitivity
- All samples negative

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

- Retroviral vector
- Stable integration, potential long-term expression
- Excellent safety record
- Transduction resulted in therapeutic FVIII levels  in preclinical models
- Intravenous delivery
- Non-invasive (hand vein)
-  patients prone to bleeding, liver dysfunction
- Targets cells in direct contact with blood
- secreted FVIII must bind to vWF and circulate
-  multiple tissues can produce FVIII

## Derived from Moloney Murine Leukemia Virus
Replication deficient
Carries gene for human Factor VIII protein 
Human complement-resistant
Manufactured at high titer (108-109 TU/ml) 

- Replication deficient
- Carries gene for human Factor VIII protein
- Human complement-resistant
- Manufactured at high titer (108-109 TU/ml)

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

## Preclinical Biolocalization Studies Using PCR to Detect Vector Sequences 

- 122 rabbits, 4 hemophilic dogs received I.V. vector
- Tissue samples obtained at different timepoints after infusion
- PCR assay with single copy sensitivity
- Multiple replicates tested  (1g DNA/replicate)
- ~ 150,000 diploid genome equivalents/replicate
- Statistical assumptions
- Reactions independent of each other
- Sampling representative of the vector distribution in the specimen

## Summary of Biolocalization Studies in Rabbits and Hemophilic Dogs

- Localization of vector sequences after IV injection:
- 	- liver / spleen			high signal persists to 2 years
- 	- bone marrow / PBMC		high signal declines over time
- 	- lung / thymus / kidney 		intermittent, threshold signal
- 	  lymph node / testis
- 	- brain				negative
- 	- semen				negative in dogs (weeks 22 and 98)                     					and rabbits (through 21 weeks)

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

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

**Time after vector infusion (days)**

**Probability of a transduced cell**

**0.000020**

**0.000015**

**0.000010**

**0.000005**

**0.000000**

**15**

**727**

**4**

**90**

##        Semen Analysis Study Design

- Adult male rabbits (N=13) treated with hFVIII(V)
- Semen samples collected weekly for PCR testing
- Duration: 21 weeks to span multiple cycles of spermatogenesis
- Clinically-relevant doses given I.V. to ensure
- therapeutic hFVIII levels in blood
- positive PCR signal in testis

## Results of Rabbit Semen Study 

- No positive semen samples
- For each sample: 20 replicates, 2 primer sets
- Incidence of sporadic, unconfirmed signals
- untreated controls =  0.00125  (1/800)
- treated animals =   0.00070 (3/4281)
- Thus, false positive PCR rate = 0.078%
- lower than published rates from other labs

## PCR Results in Rabbit Testes and Semen

- Testes
-    After one cycle of spermatogenesis (90 days), risk of 	any single rabbit testis cell containing the vector 		genome was 1 in 937,000 (1 in 709,000, 99%  confidence limit).
- Semen
-    All samples negative throughout weekly testing over multiple cycles of spermatogenesis

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

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

- Endpoints: Safety, circulating FVIII levels
- Study design:
- Single treatment - IV infusions, days 1-3
- Dose escalation (2.7 x 107 to 8.8 x 108 TU/kg)
- Subjects: Adult men with severe Hemophilia A
- Informed consent: barrier contraception; risk of IGT
- Semen test points: pre-infusion, weeks 2, 6, 9, 11, 17, 29, 53 (plus additional samples in case of positive result)
- Duration: one year

## Current Study Status

- 12 subjects on study for at least 3 months
- 4 dose levels administered, 3 subjects at each
- Infusions well-tolerated
- All subjects alive, in their usual states of health
- Semen PCR results available from
-  11 subjects through week 17
-   8 subjects through week 29
-   3 subjects through week 53

## Clinical PCR Assay for Vector in Semen  

- 10 replicates tested per sample, 1 g DNA/replicate
- Single copy sensitivity
- Assay procedures designed to minimize common sources of contamination leading to false positives
- Collections scheduled to maximize chance of detecting transduced sperm

## Spermatogenesis Model Supporting the Semen Testing Protocol

- Germ cells accessible to blood-borne RVV must be dividing and on blood side of Sertoli-cell barrier
- Differentiating spermatogonia (many/rapidly dividing), dividing stem cells (few)
- NOT spermatocytes, spermatids, or spermatozoa
- Timing of transduced sperm in semen is predictable
- Interval based upon time required for maturation from transduced precurser
- 49-92 days after vector infusion (study weeks 8-14)

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

**Vector infusions**

**Wk 18**

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

**Last sperm in semen from first cycle of spermatogenesis**** **

**0**

**Peak appearance**** **

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

**Wk 0**

**Wk 2**

**Wk 6**

**Wk 9**

**Wk 11**

**Wk 17**

## Human Semen PCR Results to Date

- 63 semen samples tested from 11 subjects
- Results: 	61 negative
- 1 positive (1/10 replicates tested)
- 1 indeterminate (contamination suspected)

## Semen Test Results  Subject #01001 / F8V101   

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

**Vector infusion**

**Wk 18**

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

-  **Last sperm in semen from first cycle of spermatogenesis**

**0**

**Peak **
**appearance**

**Wk 0**

**Wk 2**

**Wk 6**

**Wk 9**

**Wk 11**

**Wk 12**

**Wk 14**

**Wk 17**

## Possible Sources of Positive PCRTest Result in Subject #01001

- Transduced somatic cell
- Semen contains PMNs, macrophages, lymphocytes, epithelial cells
- Subject’s PBMC are PCR-positive for vector
- Test contaminant
- Sperm produced from transduced spermatogonia
- Low frequency - 1 positive out of 10 replicates
- Detected during first cycle of spermatogenesis
- Repeat samples negative

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

**1 / 190**** **

**One in 57,000,000**

**No. Positive/**
**No. Replicates Tested**

**Probability of a Sperm Cell Being Transduced**

**Pooled high risk data - weeks 9 & 11**** **

**1 / 70**** **

**One in 21,000,000**

** ****One in**
**8,600,000**

**99% Confidence Bound**

**One in 3,100,000 **

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

*******Highest dose completed to date**
**  ****= 4.4 x 10****8**** TU/kg**

## Results of Human Semen PCR Analysis 

- 	At a 99% confidence level, the worst case frequency of a transduced sperm is:
- 			1 in 3,100,000 - 8,600,000 cells
- 			or  0.0000003  to 0.0000001
- No positive samples have been detected after the first cycle of spermatogenesis

## Conclusions

- Current human data in semen is consistent with preclinical data: the probability of a germline cell being transduced is very low.
- The probability of inadvertent germline transmission of this retroviral vector genome at the current dose is remote.

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

- Repeat tests negative x 3 over three months and negative at study end (1 year)
-  No additional semen collection required
- Repeat tests positive, or sporadically positive, after first 3 months and to study end
- Perform WBC/sperm cell separation, if feasible
- Estimate risk of germ line transmission based on signal frequency
- Provide genetic counseling re. possible outcomes of conception and reproductive alternatives

## Chiron   
Vector R & DNicholas DePolo, Carlos Ibanez,  Douglas Jolly, Sybille Sauter
Vector ProductionK. Jon Kowal, James Marich, Holger Roehl, Paula Stemler 
PreclinicalMartin Giedlin, Martha Leibbrandt, Joanne Rose Layshock
ClinicalVeronica Cole, Deborah Hurst, 
	Biao Lu 
Development Donald Gay, Biff Owen 

**Vector R & D**Nicholas DePolo, Carlos Ibanez,  Douglas Jolly, Sybille Sauter
**Vector Production**K. Jon Kowal, James Marich, Holger Roehl, Paula Stemler
**Preclinical**Martin Giedlin, Martha Leibbrandt, Joanne Rose Layshock
**Clinical**Veronica Cole, Deborah Hurst,
- 	Biao Lu
**Development** Donald Gay, Biff Owen

***Clinical Investigators***
-   Jeanne Lusher MDWayne State U. Medical Center,  Detroit
-    Jerry S. Powell MDU. California Davis Medical Center, Sacramento
-    Margaret Ragni MD, MPHU. Pittsburgh Medical Center, Pittsburgh
-    Gilbert White MDU. of North Carolina Medical Center, Chapel Hill

**Contributors - FVIII Study**

***Consultant**** *
-     Kim Boekelheide, MD, PhD
-      Brown U., Providence