Preclinical Animal Models in Gene Transfer Research Anne M. Pilaro, Ph.D. FDA/CBER Biological Response Modifiers Advisory Committee Bethesda, Maryland

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 Model DiseaseMRL/lpr mice Lupus

W/Wv 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

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


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


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


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


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


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


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


Preclinical efficacy data in hemophilic mice factor VIII knock-out mouse model (C57BL/6) 2.4 x 1011 to 8 x 1012 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


Preclinical toxicity in normal mice 4 x 1010 to 4 x 1012 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 1011 vp/mouse miniAd-FVIII


Preclinical toxicity in cynomolgus monkeys 2 monkeys per group; PBS control 4.3 x 1011, 1.4 x 1012, 4.3 x 1012 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 1012 vp/kg miniAd-FVIII


Parameter Mouse Monkey

FVIII (mU/ml) 202-490a 88, 24

ALT (U/L) 68, 141b 55, 53

Plts(1000/mm3)

44 15, 34

NOAEL 1.6 x 1012 vp/kg 1.4 x 1012 vp/kga data from pharmacology studiesb n = 2; sample hemolyzedGenstar Therapeutics, used by permission

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


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

cells


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


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


Species Apparent NOAEL NOAEL (pfu/m2)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 109 IU, or 1.2 x 109 IU/m2


redesigned trial smaller volume, E1, E3-deleted vector (second

generation) “spray gun” approach single and repeat administration dose cohorts


clinical data show: single administration tolerated up to 3 x 106 pfu

dose-related, positive gene transfer at high doserepeat administrations tolerated up to 2 x 109 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)


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


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

Documents

Preclinical Animal Models in Gene Transfer Research Anne M. Pilaro, Ph.D. FDA/CBER Biological Response Modifiers Advisory Committee Bethesda, Maryland