PRIMARY CELL-MEDIATED GENE TRANSFER

- Gene transfer by embryo cloning -

Jim RoblHematech, Inc.

AC21 Advisory Committee Meeting

November 28, 2007

DNA TRANSFER BY PRIMARY CELL CLONING

Vector Primary fibroblast cells Transgenic cells

Transfection Selection

Embryonic cloning

Enucleated oocyte

Primary cell – Cell with a finite lifespan

Primary Transfection andselection

Cell lineestablishment

Transfection andselection

Cell lineestablishment

Transfection andselection

MULTIPLE GENETIC MODIFICATIONS

Additional modificationsAdditional modifications

Clone toRejuvenate

Clone tomake Calves

Clone toRejuvenate

Clone toRejuvenate

Clone tomake Calves

1st Modification

2nd Modification

GENE TARGETING

STOP SEQUENCE

(GENE KNOCKOUT)SELECTION SEQUENCE

DNA Sequence(chromosome)

Gene Sequence

1st Chromosome

2nd Chromosome

Homologous arms

Recombination

PRION GENE KNOCKOUT CATTLE

GENE KNOCKOUT CATTLE

IMMUNOGLOBULIN GENE KNOCKOUT CATTLE

MICROCHROMOSOME TRANSFERMICROCHROMOSOME TRANSFER

Chromosome Chromosomefragment

GENE SEQUENCE OF INTEREST

REMOVE UNWANTEDGENES Transgenic cells

TRANSCHROMOSOMIC CALVESTRANSCHROMOSOMIC CALVES

THERAPEUTIC PROTEINS PRODUCED IN MILK

Product Company Species Developmental Stage

Anti-thrombin III GTC Biotherapeutics Goat EMEA approvedUS: Phase III

C1 Esterase Inhibitor Pharming Rabbit Phase III

Fetoprotein Merrimack & GTC Biotherapeutics Goat Phase II

-Glucosidase Pharming Rabbit Phase II (on hold)

Human growth hormone BioSidus Cattle Preclinical

Human serum albumin GTC Biotherapeutics Cattle Preclinical

Fibrinogen Pharming Cattle Preclinical

Collagen Pharming Cattle Preclinical

Lactoferrin Pharming Cattle Preclinical

-1 antitrypsin GTC Biotherapeutics Goat Preclinical

Malaria vaccine GTC Biotherapeutics Goat Preclinical

CD 137 Mab GTC Biotherapeutics Goat Preclinical

Niemann, H. & W.A. Kues. Reprod Fertil Dev 2007; 19: 762-770.

PIG-TO-HUMAN ORGAN TRANSPLANTATION

Transgenic trait Key molecule Reference

Minimize complement activation

Human complement regulatory proteins

Cozzi & White, 1995Platt & Lin, 1998Peterson et al. 2006

Minimize hyperacute rejection

1,3--galactosyltransferase

Phelps et al. 2003Kuwaki et al. 2005Yamada et al. 2005

Minimize blood coagulation

Human thrombomodulin Peterson et al. 2006

Minimize blood coagulation

Human heme-oxygenase-1

Peterson et al. 2006

Minimize viral transmission

Porcine endogenous retrovirus

Dieckhoff et al. 2006

Niemann, H. & W.A. Kues. Reprod Fertil Dev 2007; 19: 762-770.

TRANSGENIC LIVESTOCK FOR AGRICULTURE

Trait Key Molecule Species Reference

Increased growth rate; reduced fat Growth hormone Pig Nottle et al. 1999

Increased growth rate; reduced fat Insulin-like growth factor-1 Pig Pursel et al. 1999

Increased poly-unsaturated fat Desaturase (spinach) Pig Saeki et al. 2004

Increased poly-unsaturated fat Desaturase (C. elegans) Pig Lai et al. 2006

Phosphate metabolism Phytase Pig Golovan et al. 2001

Milk composition -lactalbumin Pig Wheeler et al. 2001

Influenza resistance Mx protein Pig Muller et al. 1992

Enhanced disease resistance IgA Pig, sheep Lo et al. 1991

Wool growth Insulin-like growth factor-1 Sheep Damak et al. 1996a,b

Visna virus resistance Visna virus envelope Sheep Clements et al. 1994

BSE resistance Prion protein Cattle Richt et al. 2007

Milk fat composition Stearoyl desaturase Goat Reh et al. 2004

Milk protein -casein, -casein Cattle Brophy et al. 2003

Milk protein Human lactoferrin Cattle Platenburg et al. 1994

Mastitis resistance Lysostaphin Cattle Wall et al. 2005

Niemann, H. & W.A. Kues. Reprod Fertil Dev 2007; 19: 762-770.

SUMMARY

• Technologies exist to make complex genetic modifications in livestock species

• Commercial effort focused on human therapeutic applications

• One product currently approved for marketing

• Agricultural applications at research level