U.S. Food and Drug Administration

1. U.S. Food and Drug Administration Notice: Archived Document The content in this document is provided on the FDA’s website for reference purposes only. It was current when produced, but is no longer maintained and may be outdated.

2. Summary Data from Animal Clones Eric S. Dubbin, D.V.M. Ruminant Drugs Team Center for Veterinary Medicine Food and Drug Administration

3. Critical Biological Systems Approach Cell Fusion Through Fetal Development Perinatal Development and Function Juvenile Development And Function Reproductive Development And Function Post-Pubertal Maturation Offspring

4. Presentation Goals • Overview of literature findings • Species-by-species evaluations • Key outcomes in CBSA context • Emphasis on Development Node data relevant to • Animal health risk • Food consumption risk • More detailed presentation of Cyagra data • Largest single data set • Most detailed

5. CBSA: Summary of 1st Developmental Node Cell Fusion Through Fetal Development • Period of highest risk for developing clone • Failure to divide or implant • Reprogramming defects • Placentation difficulties • Common across all species evaluated • Percentage of successful outcomes very low • Data of limited utility for further assessment of risks

6. Bovine Clones Critical Biological Systems Approach

7. CBSA: Bovine Perinatal Clones • Key studies from a few laboratories • Many address/intermingle transgenic clones • Number of “just” clones relatively small (~50) • Most information on overall health • Few physiological/biochemical studies • Large Offspring Syndrome most common

8. CBSA: Bovine Perinatal Period Summary • Newborn clones fragile during first days • Higher incidence of death early in technology development • No qualitative differences relative to other ARTs • Frequencies often increased • Most common anomalies LOS related • BW at birth often higher than comparators • Cardiovascular malformations • Respiratory difficulties • Flexor tendon contracture

9. Critical Biological Systems Approach Cell Fusion Through Fetal Development Perinatal Development and Function Juvenile Development And Function

10. CBSA: Bovine Juvenile Clones • Period of resolution of initial instabilities • ~100 clones in all the reports • 3 reported deaths in otherwise healthy-appearing clones • 1 study has  in GH and IGF-I levels in clones than controls (Govoni et al. 2002) • Growth and general health usually reported as “normal” • Behavior normal; resembles donor animal (Savage 2003)

11. CBSA: Bovine Juvenile Clones • One clone w lymphoid hypoplasia; dies at day 51 (Renard et al 1999) • Japanese Black Beef clones of the 12 clones surviving perinatal period, all were “healthy and normal” up to 350 days (Kato 1998, 2000) • 4 clones from ear cells of a 17 yr old Japanese Black Beef bull alive and healthy at 10-12 months. Vet exams, growth curves, and 30 blood parameters reported as “normal”. No data provided. (Kubota et al. 2000)

12. CBSA: Bovine Juvenile Clones • Physiological measurements on 21 healthy-appearing clones (Chavatte-Palmer et al. 2002) •  body temp w/o infection resolved by 50 days • Some blood parameters initially unstable; resolve in a few weeks • Basic clinical chemistry normal • Hormone levels: IGF-I, IGF binding protein, leptin, insulin (post-prandial glucose) all normal • Initial thyroxine, leptin, IGF-II differences resolved within 7- 15 days

13. CBSA: Juvenile Developmental Node Summary • Initial physiological instabilities resolve during this time period • Lab results –> appropriate responses to growth and development • Physical anomalies clearly detected • Normal and healthy animals behave and appear functionally indistinguishable from Comparators

14. Critical Biological Systems Approach Cell Fusion Through Fetal Development Perinatal Development and Function Juvenile Development And Function Reproductive Development And Function

15. CBSA: Reproductive Development and Function • Data quite limited • Often cursory mention of “normal activity” • Explicit evaluation of bovine clone reproductive function (Enright et al. 2002; Govoni, Savage) • Puberty somewhat later (314 d v. 272 d) • Higher BW at first estrus (336 kg v. 302 kg) • No difference in cycle length, follicular development, hormonal profiles (daily LH, FSH, E2, & P4 similar) • 3/4 clones and 4/4 controls preg post AI (# of inseminations not reported)

16. CBSA: Reproductive Development and Function • 5 live clones healthy, normal, and normally cyclic at 1yr of age(Heyman et al. 2002 ) • “Gene” the bull, healthy, fertile, sired calves (AI/IVF)Forsberg et al. (2002) • One clone bred via AI, conceived, delivered normal calfKato et al. (2000) • 2 Holstein clones Aoki et al. (2003) abstract • First post partum ovulations delayed • Follicular waves (2/cycle) normal • Calved normally

17. CBSA: Reproductive Development and Function Summary • Clones appear to develop normally • Puberty reached ~ normally, but may be slightly delayed • Reproductive function appears normal in males and females

18. Critical Biological Systems Approach Cell Fusion Through Fetal Development Perinatal Development and Function Juvenile Development And Function Reproductive Development And Function Post Pubertal Maturation

19. CBSA: Post Pubertal Maturation • Few bovine clones have reached advanced age • No reports of sudden abnormalities arising • Hill et al. (2000) • “Second Chance”, overcame neonatal diabetes, now 3 years of age w/ normal BW, growth, behavior, and semen production

20. Data Provided by from Cyagra, Inc. • Late Spring 2003, influx of new data from Cyagra • Physical exams • Medical records • Systematically collected laboratory data

21. Clinical Medicine • History – chief complaint • Physical Exam • Systematic approach • Done the same way each time • Systems based • Clinical Pathology • Serum Chemistry • Electrolytes • Blood Cells • Red Blood Cells • White Blood Cells • Platelets • Urinalysis • Summary – Lab Data must be in Context

22. Clinical Pathology • Complements the physical exam • Assist diagnosis for a disease process • Reference Ranges have limitations • Based on a population of healthy animals • Mean +/- 2 SD • 5% of normal values may be out of range • Lab’s reference population • Artifact • Clinical Judgment Still Necessary

23. Cyagra Data Set

24. Cyagra Health Data • Mortality • Physical Exam • Clinical & Surgery– Umbilical problems • Cull

25. Cyagra Health Data • Mortality • 134 clone calves • 28 stillborn, died, or euthanized by 48 hrs • 11 calves died from 2d to 1 yr • 8 from d2- 29 • 3 from d30-180 • 0 after 180

26. Cyagra Health Data • Mortality (of 134 clones) • 28 stillborn, died, or euthanized by 48 hrs • Stillborn – 11 • Euthanized – 12 • Abnormal development 7 • Contracture 5 • Diarrhea – 2 – rotavirus • Unknown – 3 • Appeared normal • thick placenta & pericarditis • Moist umbilicus, depressed

27. Cyagra Health Data • Mortality (of 134 clones) • 11 clones died after 2d • 3 septic umbilical problems • 3 G.I. bloat, adhesions, abomasal rupture • 3 neonatal circulatory problems • 1 contracture, non responsive • 1 accidental trauma

28. Cyagra Health Data of Surviving Clones • Physical Exams • 11 clones - musculo skeletal abnormalities • 9 clones with contracted tendons – survived • 1 thick withers, enl. carpus, leg dev. laterally – culled • Dwarf with frequent bloat, eventually culled • 1 clone - early mammary development • 2 clones - harsh lung sounds • 3 bull clones - a retained testicle (cryptorchid) all were from the same donor bull • 1 bull clone - cardiac arrhythmia

29. Cyagra Health Data • Umbilical • 41 umbilical difficulties • Bleeding, infection, adhesions, • 2 had subsequent pyelonephritis • Surgeries • 29 clones had umbilical surgery • Miscellaneous health problems • 3 clones had rotavirus • 2 clones died of that disease • 2 G.I.- bloat, adhesions, ruptured abomasum • 1 Failure to transition to fetal circulation • 1 hypoxia, rapid deterioration • Cull • 2 -1 dwarf and 1 with thick withers, enlarged carpus, deviated leg

30. Clinical Chemistry, CBC, and Urinalysis • Organization and interpretation of data • Decide on most appropriate comparison

31. Summary of Laboratory Value Comparisons

32. Clinical Chemistry, CBC, and Urinalysis • Creation of comparative chart approach • Good visual comparison • Concise, but informative

33. Chart 311: Hemogram Clones v Comparator [6-18 mo]

34. Clinical Chemistry and CBC • What questions do we want these data to answer? • Similarity/Differences • Response to internal and external stimuli • Predictability

35. Trend 1- Age Appropriate Responses in Growth Analytes • Growing animals, both clones and comparators, have elevations in growth specific analytes relative to adults • Alkaline phosphatase • Ca++ • P

36. Trend 2 – Age Appropriate Responses in Immunoglobulin • Endogenous Immunoglobulin Production • Total protein • Globulin • Young calves, both clones and comparators, have low globulin levels • Older calves have higher globulin levels

37. Cyagra Neonates: Clinical Chemistry

38. Cyagra Neonates: Hematology

39. Neonates: Summary • 10 clones, 2 died • Similarity: 92% clin chem; 90% hematology • Liver enzymes different, normalize by next node • Response • Growth factors and immune factors show expected distribution • Predictability • Dead clone with many analytes out of range • Dead clone with few analytes out of range • Laboratory values must be interpreted in the context of the animal.

40. Cyagra 1-6 Month Group: Clin Chem

41. Cyagra 1-6 Month Group: Hematology

42. Cyagra 1-6 Month Group: Summary • 46 clones with blood work, one culled • 3 died in this age group (no blood work) • Similarity to comparators: • 96% clin chem; 96% hematology • Elevated growth analytes, glucose relative to Comparators • 4 animals, same donor, different phenotypes • 3 cryptorchid, same bull donor • Response • Appropriate immune response & WBC elevation in response to abscess • Predictability • Clone had only one analyte out of range, health problems • 3 clones with many analytes out of range, no health problems • Laboratory values must be interpreted in the context of the animal.

43. 7 Clone Subcohort Analysis • Neonatal Chemistry • Colostrum • Globulin • 0/7 neonates low • 3/6 1-6mo low, reflects maternal Ig waning • Lower GGT • 4/7 neonates, pre-colostrum, normalized by 1-6mo • Liver • Lower AST • 6/7 neonates, normalized by 1-6mo • Lower Cholesterol & Bile Acids • 5/7 neonates, normalized by 1-6mo • Lower GGT • 4/7 neonates, normalized by 1-6mo

44. 7 Clone Subcohort Analysis (cont.) • Neonatal Hematology • One clone with decreased lymphocytes and platelets • Normalized by 1-6mo • #79 • Neonate • Decrease AST, decreased bile acids • Had no hematology data • Juvenile (1-6 mo) • Increased creatinine, dec glob, inc alk phos, inc cholesterol • Hematology - dec TP-ref, RDW, and inc MPV. • Clone culled for poor conformation

45. 7 Clone Subcohort Analysis (cont.) • 1-6 Month Clones • 4 elevated glucose, no glycosuria • 4 elevated alk phos, younger animals • 3 elevated P, younger animals • 3 increased A/G ratio (assoc with dec glob) • 1 elevated creatinine, only marginal, no other renal signs • 2 decreased anion gap, unknown cause These out of range values do not indicate any consistent trends other than expected physiological changes

46. 7 Clone Subcohort Analysis (cont.) • Summary • Out of range neonatal values improved by 1-6mo • Out of range 1-6mo values, when taken in context, were not of clinical concern • 1 clone later culled for poor conformation

47. Cyagra 6-18 Month Group: Chemistry

48. Cyagra 6-18 Month Group: Hematology

49. Summary: Cyagra 6-18 Month Group • 18 clones with blood work • 0 died in this age group • Similarity: • 99% clin chem; • 99% hematology • IGF-I and GH, only slightly elevated to comparator, but within published and physiologic ranges • Response no disease to monitor

50. Swine • Archer 2003 – • determine variation within a population of clones and compare to pigs of • genetically similar age, • breed (Duroc), and • sex matched naturally bred females • Half siblings