# Presentation: 587302

## Preclinical Animal Models in Gene Transfer Research

**Anne M. Pilaro, Ph.D.**
**FDA/CBER**
**Biological Response Modifiers**
**Advisory Committee**** **
**Bethesda, Maryland**
**November 16, 2000**

## Initial Steps in the Development of a New Gene Transfer Vector

**Product characterization**
- manufacturing and quality control issues
**Biologic activity**
*in vitro* and/or *in vivo* “proof of concept”
**Safety**
- toxicology testing in animals
- safety
- biodistribution

## Goals of Preclinical Bioactivity Studies in Animals

**Establish rationale for conducting trial**
- feasibility of gene transfer
- duration, levels of gene expression
- degree of functional correction
**Optimize dosing, regimen for the clinic**
**Optimize route of administration**
**Selection of species for further testing**

## Goals of Preclinical Toxicology Studies in Gene Transfer Research

**Recommend initial safe starting dose and safe dose-escalation scheme in humans**
**Identify potential target organ(s) of toxicity**
**Identify appropriate parameters for clinical monitoring**
**Identify "at risk" patient populations (inclusion/exclusion)**

## Goals of Biodistribution Studies in Gene Transfer Research

**Designed to address two issues:**
**dissemination of vector to the germline**
**total gonadal tissue assayed to date**
**distribution of vector to non-target tissues**
**provides information on potential target organs for toxicity**
**both issues ****may**** be addressed in same preclinical study**

## Questions to be Answered by Preclinical Pharm/Tox Studies

**1.    What is the relationship of the dose to the ****	****	****biologic activity?**
**2.     What is the relationship of the dose to the ****	****	****toxicity?**
**3.      Does the route and/or schedule affect ****	****   ****	**** activity/toxicity?**
**4.      What risks can be identified for the ****	****	****	**** clinical trial?**

## How are Preclinical Animal Data Relevant to Gene Transfer?

**The Bottom Line...**
**   ****Prior to the availability of human data, preclinical toxicology and pharmacology studies provide the **_**sole**_** **_**source**_** of data upon which safety assessment is made**

## Animal Models are Useful to Study Potential Gene Transfer Vectors

***Animal Models are Useful to Study Potential Gene Transfer Vectors***

_**Animal Model **_**                       ****	**_**Disease**_
**MRL/*****lpr*****  mice****	****	****	****    ****	****Lupus **
**W/W****v**** mice****	****	****	****    ****	****Fanconi’s anemia**
**Wobbler mice****	****	****	****    ****	****ALS**
**db/db mice****	****	****	****    ****	****Diabetes**
***spf***** mouse****	****	****	****	****    ****	****OTC deficiency**
***mdx *****mice****	****	****	****	****    ****	****Muscular dystrophy**
***cftr*****-KO mouse****	****	****	****    ****	****Cystic fibrosis**
**Watanabe rabbit****	****	****	****    ****	****Hypercholesterolemia**
**hemophilic dogs****	****	****	****    ****	****Hemophilias A/B**
**various MPS defects (dog, cat, rat)    ****	****Lysosomal storage**

## Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer 

***Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer ***

**the disease - metastatic breast cancer**
**the gene defect - unknown; may include abnormal mutations in BRCA-1, c-*****fos*****, p53, others**
**clinical outcome**
**aggressive, spread through lymphatics**
**metastases to lung, chest wall, peritoneum**
**poor response to chemotherapeutics**
**median survival <40% for 5 years**

## Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer

***Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer***

**gene therapy approach:**
**retroviral vector, expressing MDR-1 gene**
**CD34+ stem cells transduced *****ex vivo***** with vector, reinfuse into patients after HDCT**
**MDR-1 expression by stem cells confers resistance to taxol, other marrow-toxic agents**
**reconstitution by MDR-1 positive marrow allows escalating doses of taxol, increased anti-tumor effect**** **

## Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer

***Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer***

***In vitro***** “proof of concept” studies**
**positive gene transduction, resistance to taxol in both human, murine stem cells**
**higher level of gene transfer observed in murine than human stem cells**** **
**no inhibition of cell proliferation, altered phenotype by FACS**

## Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer

***Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer***

***In vitro***** “proof of concept” studies, cont’d**
**7 to 11% increase in Rh123 efflux in transduced human cells by FACS (index of MDR-1 function)**
**Hegewisch-Becker *****et al*****.,  *****Brit. J. Haematol.*****, **_**90**_**:1876-883, 1995**
**Hanania *****et al.*****, *****Gene Ther*****., **_**2**_**:285-294, 1995**
***In vivo***** studies**
**serial bone marrow transplants with MDR-1 transduced cells, escalating taxol doses in mice **

## Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer

***Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer***

***Safety Issue*****: Will transfer and/or expression of MDR-1 gene affect engraftment/reconstitution, function of marrow cells?**
**no formal toxicology testing was conducted for this system**
** ****HOWEVER, monitored mice during efficacy study for toxicity endpoints**
**Hanania *****et al.*****, *****Cancer Gene Ther*****, **_**2**_**: 251, 1995**
**Hanania and Diesseroth, *****Clin Cancer Res, ***_**3**_**:281, 1997**

## Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer

***Case Study - Using an Efficacy Model to Support Safety of Gene Transfer for Cancer***

***Safety Issue*****: Will transfer and/or expression of MDR-1 gene affect engraftment and reconstitution, function of marrow cells?**
**no adverse effects on engraftment, reconstitution**
**beneficial effect on survival**
**conferred resistance to escalating doses of taxol**
**support from literature that transgenic MDR-1 mice have no adverse effect on engraftment**
**Mickisch *****et al*****., *****Proc Natl Acad Sci USA*****, **88**:547, 1991**

## Case Study - Comparison of Vector Toxicity in Monkeys and Mice

**the disease - hemophilia A**
**the gene defect - mutation in factor VIII gene**
**defective/absent factor VIII  (FVIII) production**
**clinical outcome**
**X-linked disease; affects males only **
**severe  deficiency (< 1% normal FVIII) associated with increased morbidity**
**uncontrolled bleeding events require exogenous FVIII**
**repeated hemarthrosis leads to significant joint disease**

## Case Study - Comparison of Vector Toxicity in Monkeys and Mice

**gene therapy approach:**
**“****mini”adenovirus vector, carrying FVIII cDNA**
** ****instill by i/v infusion to target liver**
**vector contains liver-specific promoter sequence**
** ****monitor for gene presence, expression by plasma FVIII levels (ELISA)**
**correction of FVIII levels to as low as 1% to 5% of normal can lead to improved phenotype**

## Case Study - Comparison of Vector Toxicity in Monkeys and Mice

**Preclinical efficacy data in hemophilic mice**
**factor VIII knock-out mouse model (C57BL/6)**
**2.4 x 10****11**** to 8 x 10****12**** vp/kg miniAdFVIII, i/v**
**detectable FVIII in plasma out to 247 d **
**peak FVIII levels 164 to 892 ng/ml**
**normal level in humans 200 ng/ml**
**restoration of clotting time to normal limits**
**similar results in normal, C57 or BALB/c mice**

## Case Study - Comparison of Vector Toxicity in Monkeys and Mice

**Preclinical toxicity in normal mice**
**4 x 10****10**** to 4 x 10****12**** vp miniAdF-VIII, i/v**
**Toxicities**
**transient, dose-rel decrease in platelets, d 4**
**transient, dose-rel increase in ALT, d 4**
**minimal microscopic pathology in liver**
**NOAEL = 4 x 10****11**** vp/mouse miniAd-FVIII**

## Case Study - Comparison of Vector Toxicity in Monkeys and Mice

**Preclinical toxicity in cynomolgus monkeys**
**2 monkeys per group; PBS control**
**4.3 x 10****11****, 1.4 x 10****12****, 4.3 x 10****12**** vp/kg, i/v**
**Toxicities**
**transient, dose-rel decrease in platelets, d 3**
**transient, dose-rel increase in ALT, d 3**
**minimal toxicity in liver on histology**
**NOAEL = 1.4 x 10****12**** vp/kg miniAd-FVIII**

## Case Study - Comparison of Vector Toxicity in Monkeys and Mice

## Case Study - How do Safety Data in  Animal Models Compare to Humans? 

**the disease - cystic fibrosis**
**the gene defect - mutation in CFTR gene**
**defective Cl****-**** secretion, clearance in lungs**
**clinical outcome**
**chronic respiratory infections**
**chronic digestive disorders**
**sterility in males**
**death by age 35-40**

## Case Study - How do Safety Data in  Animal Models Compare to Humans? 

**gene therapy approach:**
**adenovirus vector, carrying CFTR**
**instill through bronchoscope to one lobe of lung**
**monitor for gene presence by brush biopsy of cells **

## Case Study - How do Safety Data in  Animal Models Compare to Humans? 

**animal models not appropriate for pharmacology or efficacy**
**CFTR-ko mouse has digestive disease, no lung pathology**
**preclinical safety studies showed dose-limiting inflammation in lungs**
**all species tested, rodent or non-human primate**
**dose-related, sharp threshold**

## Case Study - How do Safety Data in  Animal Models Compare to Humans? 

- clinical data show:
- no toxicities at initial doses of 2 x 107 pfu virus, instilled
- dose-limiting, inflammatory lung reaction at 2 x 109 pfu
- had to stop initial trial due to toxicity
**Crystal, R.G. *****et al.*****, *****Human Gene Ther.*****, 6:643-666, 1995**

## Case Study - How do Safety Data in  Animal Models Compare to Humans? 

- _Species__	__	__Apparent NOAEL__	__NOAEL (pfu/m_2_)_
- C57 BL/6		2.6 x 107 pfu/mouse	2.4 x 109 pfu/m2
- hamster		3.6 x 107 pfu/hamster	1.7 x 109 pfu/m2
- cotton rat		5 x 107  pfu/rat		1.9 x 109 pfu/m2
- Rhesus monkey	2 x 107 pfu/monkey*	8.2 x 107 pfu/m2
- baboon		7 x 108 pfu/monkey	1.8 x 109 pfu/m2
- human		2 x 107 pfu/patient	1.2 x 107 pfu/m2**
***NOAEL not available; lowest dose tested with minimum pathology**
****toxic dose in humans by this route, 2 x 10****9 ****IU, or 1.2 x 10****9 ****IU/m****2**

## Case Study - How do Safety Data in  Animal Models Compare to Humans? 

**redesigned trial**
**smaller volume, E1, E3-deleted vector (second generation)**
**“****spray gun” approach**
**single and repeat administration dose cohorts**

## Case Study - How do Safety Data in  Animal Models Compare to Humans? 

**clinical data show:**
**single administration tolerated up to 3 x 10****6**** pfu**
**dose-related, positive gene transfer at high dose**
**repeat administrations tolerated up to 2 x 10****9**** pfu per dose**
**gene transfer positive after second, not third repeat dose**
**Harvey, BG *****et al.*****, *****J. Clin Invest..*****,  104:1245-1255, 1999**

## Summary

**Safety data can be obtained in efficacy models**
**Mouse studies can provide similar info as studies conducted in monkeys**
**No one species may be predictive of toxicities in humans**
**not all toxicities may be seen in all species**
**humans may not be predictive of other humans**

## Questions for the Committee

**Q1. ****When is it appropriate to require safety studies of gene transfer agents in non-human primates?**
-   In discussing this question, please consider the following:
- a).  phase of clinical trial/product development
- b).  clinical indication
- c ).  class of vector
- d ).  level of gene transfer
- e).  the limitations in terms of study design(s)

## Questions for the Committee

**Q2.  When is it appropriate to obtain these data in rodent and/or other small animal models?**
-    In discussing this question, please consider the following:
- a).  clinical indication
- b).  class of vector
- c).   what is known about the immunobiology of the vector, as well as the host immune response in the rodent vs. non-human primate models

## Questions for the Committee

- Q3.   **Should safety data in efficacy models be required for all new gene transfer protocols, prior to entry in phase 1 clinical trials?**