Title of Invention

A MODIFIED POPULATION OF CELLS DIFFERENTIATED FROM PRIMATE PLURIPOTENT STEM (pPS) CELLS

Abstract The present invention relates to a method of producing a population of differentiated cells, comprising: a) providing a cell population comprising undifferentiated stem cells that contain a nucleic acid molecule comprising the structure P-X, wherein X is a nucleic acid sequence encoding a product that is lethal to a cell in which it is expressed, or renders a cell in which it is expressed susceptible to a lethal effect of an external agent, and P is a transcriptional control element that causes X to be preferentially expressed in undifferentiated cells; and b) causing at least some undifferentiated cells in the population to differentiate.
Full Text

DIFFERENTIATED CELLS SUITABLE FOR HUMAN THERAPY
TECHNICAL FIELD
This invention relates generally to the field of cell biology of embryonic cells, and the molecular biology of prcmoter controlled viral vectors. More specifically, it describes a lechnoiogy for removing undifferenîiated cells from populations derived from pluripotent stem cells using selectively expressed lytic vectors.
REFERENCE TO REUTED APPLICATIONS
This application daims priority to U.S. Patent Applications 60/253,443 and 60/253,357, filed November 27, 2000, pending; and 09/783,203, filed February 13, 2001. pending. For purposes of prosecutlon of this application in the U.S., the priority documents are hereby incorporated herein by référence In their entirety.
BACKGROUND
Precursor cells hâve become a central interest in médical research, Many tissues in the body hâve a bacic-up réservoir of precursors Ihat can replace cells that are senescent or damaged by Injury or disease. Considérable effort has been made recentiy to isolaîe precursors of a number of différent tissues for use In regenorative medicine.
U.S. Patent 5,750.397 (Tsul U.S. Patent 5,716,411 (Orgill et ai.) proposes regenerating skin at the site of a burn or wound, using an epilheliai aulograft. U.S. Patent 5,766,948 (F. Gage) reports a method for producing neurobiasts from animal brain tissue. U.S, Patent 5,672,499 (Andersen et al.) reports oblaining neurai crest stem cells from embryonic tissue. U.S. Patent 5,851.832 (Weiss et al., Neunospheres) reports isolation of putative neurai stem cells from 8-12 week old human fetuses. U.S. Patent 5,968,829 (M. Carpenter) reports human neurai stem cells derived from primary central nervous System tissue.
U.S. Patent 5,082,670 (F. Gage) reports a method for grafting genetically modified cells to treat defects. disease or damage of Ihe central nervous System. Auerbach et aJ. (Eur. J. Neurosci. 12:1696, 2000) report that multipotential CNS cells implanted into anima! brains form eleclrically active and functionally connected neurons. Brustle et al. (Science 285:754,1999) report that precursor celis derived from embryonic stem cells interact with host neurons and efficientiy myeiinate axons in the brain and spina! cord.
Considérable interest has been generated by the development of embryonic stem celis, which are thought to hâve the potentiai to differentiate into many cell types. Early work on embryonic stem cells was done in mice. Mouse stem cells can be isolated from both early embryonic cells and germinal tissue. Désirable characteristics of pluripotent stem cells are that they be capable of prolifération in vitro in an undifferentiated staîe, retain a normal karyotype, and retain the potentiai to differentiate to derivatives of all three embryonic germ layers (endodenn, mesodenn, and ectoderm).

Development of human pluripotent stem cell préparations is considerably iese advanced than work with mouse cells. Thomson et a!, propagated pluripotent stem cells from lower primates (U.S. Patent 5.843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995), and then from humans (Science 282:114, 1998). Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal çonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726,1998; and U.S. Patent 6,090.622).
Both hES and hEG cells hâve the long-sought characteristics of pluripotent stem cells: they are capable of being grown In vitro without differentiating, they hâve a normal karyotype, and they remain capable of producing a number of différent cell types. Clonally derived human embryonic stem cell lines maintain pluripolency and proliferative potentiel for prolonged periods in culture (Amit et a!., Dev. Bîol. 227:271, 2000). Thèse cells hold considérable promise for use in human therapy, acting as a réservoir for régénération of almost any tissue compromised by genelic abnormality, trauma, or a disease condition.
international Patent Publication WO 99/20741 (Geron Corp.) refers to melhods and materials for growing primate-derived primordial stem cells. In one embodimenl, a cell culture médium is provided for growing primate-derived primordial stem cells In a substantially undifferentiated state, having a low osmotic pressure and iow endotoxin levels. The basic médium is combined with a nutrient sérum effective to support the growth of primate-derived primordial stem cells and a substrate of feeder cells or an extracellular matrix component derived from feeder cells. The médium can further Include non-essential amino acids, an anti-oxidant, and growth factors that are either nucleosidea or a pyruvate sali.
A slgniflcanl challenge to the use of stem cells for therapy Is lo control growth and differentiailon into the particuiar type of tissue required for treatmenl of each patient.
U.S. Patent 4,959,313 (M. Taketo, Jackson Labs) provides a particuiar onhancer séquence thaï causes expression of a flanking exogenous or recombinant gène from a promoter accompanying tho gène that does nol normally cause expression in undifferentialed ceils. U.S. Patent 5,639,618 {D.A. Gay, Pluricn Inc.) proposes a method for isolating a lineage spécific stem cal! in vitro, in which a pluripotent embryonic stem cell is transfected with a construct in which a lineage-specific genetic élément is cperably linked to a reporter gène, culturing the cell under conditions where the cell differenliates. and then séparation of cells expressing the reporter are separated from other cells.
U.S. Patent 6,087,168 (Levesque et al., Cedars SInai Med. Ctr.) Is directed to transdifferentiating epldenmal cells into viable neurons useful for both cell therapy and gène therapy. Skin cells are transfected with a neurogenic transcription faclor, and cuttured In a médium containlng an antisense oligonucleotlde corresponding to a négative regulator of neuronal dlfferentiation.
International Patent Publication WO 97/32025 (Mclvor et al., U. Minnesola) proposes a melhod for engrafting drug résistant hematopoietic stem cells. The cells in the graft are augmented by a drug résistance gène (such as metholrexate résistant dihydrofoiate reductase), under control of a promoter functional in stem cells. The cells are administered into a mammal, which is then trealed with the daig to increase engraftment of transgenic cells relative to nontransgenic ceils.
Internationa! Patent Publication WO 98/39427 (Stein et al., U. Massachusetts) refers to methods for expressing exogenous gènes In differentiated cells such as skelelal tissue. Stem cells (e.g., from bone marrow) are contacted with a nuclelc acid in which the gène is linked to an élément that controls expression in differentiated cells. Exemplary is the rai osleocalcin promoter. International Patent Publication WO 99/10535 (Liu et ai., Yale U.) proposes a process for studying changes In gène expression in stem cells. A gène expression profile of a stem cell population is prepared, and then compared a .gène expression profile of differentiated cells

International Patent Publication WO 99/19469 (Braetscher et al.. Blotransplant) refers to a method for growing pluripotent embryonic stem cells from the pig. A selectable marker gène is Inserted Into the cells so as to b© regulated by a control or promoter séquence in the ES cells, exemplified by the porcine OCT-4 promoter.
International Patent Publication WO 00/15764 (Smith et al., U. Edinburgh) refers to propagation and dérivation of embryonic stem cells. The cells are cultured in the présence of a compound that seleclively inhibits propagation or survivaj of cells other than ES cells by inhibiting a signaling pathway essentîal for the differentiated cells to propagate. Exemplary are compounds that Fnhibit SHP-2, MEK, or the ras/fMPK cascade.
Klug et al. (J. Clin, Invest. 98:216, 1996) propose a strategy for genetically selecting cardiomyccvles from difîerenîiating mouse embryonic stem cells. A fusion gène consisting of the a-cardiac myosin heavy chain promoter and a cDNA encoding amînoglycoside phosphotransforase v;as stably Iransfecled into the ES cells. The resulting Unes were differentiated in vitro and selected using G418. The seîecîed cardiomyocyte cultures were reported to be hlghly differentiated. When engrafted back into mice, ES-derived cardiomyocyte grafts were détectable as long as 7 weeks after implantation.
Schuldiner et al. (Proc. Natl. Acad. Soi. USA 97:11307, 2000) report the effects of eight growth factors on the differentiation of cells from human embryonic stem cells. After initiating differentiation through embryoid body formation, the cells were cultured in the présence of bPGF, TGF-βl. activin-A, BMP-4, HGF, EGF, PNGF, or retinoic acid. Each growth faclor had a unique efîect on the differentiation pathway. but none of the growth factors direcled differentialion exclusively lo one celi type.
There is a need for new approaches to generate populations of differentiated cells suitable for human administration.
SUMMARY OF THE INVENTICN
This invention provides a System for depieting relalively undifferentiated cells from a heterogeneous cell population, such as may be obtained by differentialion of stem cells. The population is treated with a vector that puts a lethal or potentially lethal effeclor gène under control of a gène élément that ailows the gène to be expressed al a higher level in the undifferentîated subpopulation. This prcduces a population relatlvely enriched for mature cells, and suitable for use in regenerative medicine.
One embodiment of this invention is a population of cells differentiated from stem cells cultured ex vivo, v/hich is essentially free of undifferentîated cells. Exemplary are pluripotent stem cells of primate origin, such as human embryonic stem cells.
Cells In the population can contain or be derived usIng a poIynucleotide comprising the structure P-X, where X is a nuclelc acId séquence that is lethal to a cell in which it is expressed, or renders a cell in which il is expressed susceptible to a lethal effecl of an exlernal agent; and P is a transcriptional control élément that causes X to be preferentially expressed in undifferentîated cells. The Connecting Une in P-X indicales that the genetic éléments are operatively linked, whether or not they are adjacent in the nuclelc acid molécule.
X is referred to in the description that follows as an effector séquence. X can encode a toxin, a protein that induces or médiates apoptosis, or an enzyme (such as thymidine kinase) that converts a prodrug (such as ganciclovir) to a compound Ihat is lethal to a cell in which X is expressed. Other examples are provided later in this disclosure.
In certain embodiments, P-X is an introduced heterologous molécule, meaning that the cell or its ancestors was genetically altered with a vector comprising P-X. In oîher embodiments the cell or ils ancestors was genetically altered with a vector to place X under control of an endogenous transcriptional control élément.

FoIIowing transfection, X can be either transiently expressed in undifferentiated celfs in the popufation, or P-X can be inheritable and expressed in undifferentiated progeny. Non-limiting examples for P include the OCT-4 promoter, and the promoter of lelomerase reverse transcriptase (TERT). The cells can also contain a drug résistance gène Y under control of P, depicted in this disclosure as P-X-Y, indicating a functional relationship where P régulâtes transcription of both X and Y, with the éléments being in any orientation in the séquence that links the functions in this manner.
Another embodiment of the invention Is a stem cell genetically aitered so as lo contain a nudeic acid v/ith the slnjcture P-X, as aiready descrlbed. The invention aIso provides poiynucleotide vectors adapted ta genetically aller stem cells in this fashion.
Another embodiment of the invention is a method of producing a population of differentiated cells. A CEII population comprising undifferentiated stem .cells that contain a nudeic acid molécule comprising the stuccture P-X is treated to cause at least some undifferentiated cells in the population lo differentiate.
Another embodiment of the invention is a method for depietîng undifferentiated stem cells from a ceil population. Stem cells in the population are genetically aitered so that they contain a nudeic acid molécule comprising the structure P-X as aiready described. In this way, a gène that Is lethal to a celi in which- it is expressed, or rende rs it susceptible lo a lethal effect of an external agent, la placed under control of a Iranscriptional control élément that causes the geneto be preferentlally expressed In undifferentiated celis. The cell population can be genetically aitered when it is still predominantly undifferentiated (before being caused lo differentiate), or when it aiready predominantly comprises differentiated cells.
if X is lethal to the cell, then undifferentiated stem cells can be depleted simply by cuituring the cell population under conditions where X is expressed. If X rendors the cell susceptible to lethal effects of an external agent (such as a drug or prodrug), then undifferentiated stem cells are depleted by combinîng ihe cells with the external agent, This can be done by ccntacling the cells in vitro with the agent in tissue culture, cr administering the celis to the subject simultaneously or sequentially with the external agent, if not aiready présent.
The reagents and techniques of this invention can be brought to bear on cell populations containing any type of stem cells. They are especially suited for application lo primate plurlpotent stem cells, such as human embryonic stem cells.
Olher embodiments of the Invention will be apparent from the description that (ollows.
BRIËF DESCRIPTION OF THE DRAWINGS
Figure 1 provides an analysis of OCT-4 and hTERT expression In hES cells cultured with feeder cells (mEf^ or extracellular matrix (MatrigeliS) or laminln) wIth regular médium (RM) or condïîioned médium (CM). The upper panel Is a copy of a gel showing OCT-4 and hTERT expression al the mRNA level by RT-PCR. The lower panel ]s a bar graph comparlng the level of expression for cells grown on différent subslrates, expressed as the ratio of OCT-4 or hTERT lo the 18s standard. hES cells grown on Laminin and Matrigel® in conditioned médium hâve similar expression pattems to those of cells grown on a feeder layer.
Figure 2 is a half-tone reproduction of a gel showing îelomerase activity measured in cultured hES cells by TRAP activity assay. Ail the culture conditions showed positive telomerase activity afîer 40 days in feeder-free cutture.
Figure 3 is a haif-tone reproduction showing expression of the 6FP reporter gène in hES cells transduced with retrovirus and then differentiated. hES cells were transferred to suspension culture to form embryoid bodies, cultured for a further 4 days, replated onto gelatin-coated slides and cultured for a week, and

then fixed and photographed under fluorescence for GFP expression. Left panels show bright-field illumination; right panels show fluorescence due to GFP expression.
Figure 4 shows the results of a study in which hES cells were transiently genelically altered in feeder-free culture by lipofection. Panel A is a half-tone reproduction of a light micrograph showing nnorphology of hES cells on laminin after they hâve been transfected. Panel S is a half-tone reproduction of a fluorescence micrograph showing GFP expression in the same colony. Panel C is a bar graph showing percentage of ceiis expressing GFP under various conditions.
Figure 5 is a map of TPAC vector designated pGRN376. This is an adenovirus vector of 7185 bp comprising the herpès simplex thymidine kinase (ik) gène under conlrol of a promoler taken from the upstream séquence of the human gène for telomerase reverse transcriptase (hTEHT). Expression ol îk is promoled in cells expressing hTERT, such as undifîerentiated embryonic stem cells.
Figure 6 is a two-panel lina graph. showing the effect of the TPAC thymidine kinase vector on undifferentiated hES cells. 48 h after repiating, the cells were transduced with TPAC vector at an MOI cf 30 or 100, or mock transduced (no vector added). Four h later, the cells were exchanged into fresh médium containing the prodrug ganciclovir (GCV). By day 3, wells treated with TPAC vector + GCV contained 8% as many cells as the control wells.
Figure 7 is a bar graph showing titratlon of GCV In TPAC vector treated hES cells. 4 h after transduction with the vector, fresh médium was added containing GCV at the concentration shown. *20 \ihA GCV was optimal under the conditions tested.
Figure 8 Is a two-panel bar graph showing titration of GCV on TPAC vector transduced and mock-transduced hES cells from tvo différent Unes. Both lines are sensltive to GCV after treatment with the TPAC vector.
Figure 9 shows the effect of TPAC + GCV treatment on mixed cell populations obtaîned from differentialion of hES cells. The cells were fed daily with conditioned médium to malntain the undifferentiated State, or with either 500 nM retînoic acid or 0.5% DMSO, to înduce differentialion into commitled cells of mixed phenotype. 7 days later, they were infecled with the TPAC vector at an MOI of 30, plus 20 µM GCV.
The Upper Panel is a bar graph showing the number of cells surviving In culture. Treatment with TPAC + GCV eliminated cells cultured under each condition. In each instance, culture of the surviving cells produced populations that appeared highiy differentîated and substantlally free of undifferentiated morphology. The Lower Panel is a half-lone reproduction of a gel showing RT-PCR analysis of the surviving cells. Those cells cultured with conditioned médium (mEF-CM) or DMSO had no détectable OCT-4 expression, whiîe 2 out of 4 samples treated with retinoic acid (RA) showed amplification products consistent v/ith very low levels of OCT-4 expression.
Figure 10 is a reproduced micrograph of an hES cell line that has been transduced by combining with a control adenovirus vector (Panel A), or pGRN376 (Panel B). which contains the tk gène under control of the TERT promoler. Both wells of transduced cells were cultured for 3 days in a médium containing ganciclovir. Undifferentiated colonies typîcat of normal hES cell cultures were seen In the conlrol wells. In the wells treated with pGRN376, most or ail undifferentiated ES cell colonies were gone, and only differentîated cells remained.
Figure 11 Is a two-panel line graph, showing drug sensitivity of undifferentiated cells containing the telomerase promoter driven thymidine kinase gène (TPAC). Upper and lower panels shov; sensitivity io the prodajgs ganciclovir (GCV) and (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), respectively. Ganciclo'vir at a concentration as low as 2.5 µM kills virtually ail of the undifferentiated TPAC ES cells within -4 days.
Figure 12(A) and (B) comprises black-and-white reproductions of fluorescence micrographs of differentîated ES cells. Cell lines H9-376m-18, H9-376m-62. and H9-376m-6 ccntain the TPAC gène; H9-pGK-

neo-1 ts the control cell iine transfected only with the drug sélection plasmid. The stably transfected cells were differentiated into ennbryoid bodîes, and plated for inrimunocytochemistry analysis. A least three of the TPAC conlaining stem cell lines show areas that stain for muscle spécific actin, α-fetoprotein, β-tubulin, and cardiac troponin l, représentative of ail three embryonic germ layers.
DETAiLED DESCRIPTION OF THE INVENTION
stem cells of various kinds hâve become an extremely attractive modality in regeneralive mediclne. They can be.proliferated in culture, and then differentiated in vitro or in situ into the cell types needed for therapy. Recently, it has been demcnstrated that human embryonic stem cells continuously express a high level of telomerase, enabling them to maintain telomere length and grow almcst indefinitely in culture.
So far, efforts to differentiale stem cells hâve been directed primarily towards identifying culture conditions that promote outgrowth of a cell population wtih phenotypic features of a tissue type désirable for regenerative mediclne.. Schuldlner et al. (supra) report the effects of grov/th factors on the differentlatlon of human embryonic stem cells. In U.S. Patent 5,639,613, stem cells are transfected wlth a lineage-speciflc gène that is operably flnked to a reporter gène, which Is then used to sélect for cells expressing the reporter. In WO 97/32025, hematopoletic stem cells are augmented by a drug résistance gène, and then engrafted Into a subject, The cells are administered Into a mammal, which îs then treated with the drug to increase engraftment of transgente cells. Klug et al. {supra) used a construct in which the a-cardlac myosin heavy chaIn promoter controlled expression of aminoglycoside phosphotransferase. Transfected differentiated cells were selected using G418, which produced lines of cardiomyocyte llke cells. Thls Is a posith/e sélection strategy that uses gène expression patterns of the desli-ed tissue type to allow preferential survival of differentiated tissue.
It is a hypothesis of this invention that some of the populations of differentiated cells produced using adaplive culture and positive sélection melhods will be suboptimal for use in human therapy. !n some circunistances, undifferentiated cells in the population may impair engraftment or function of Ihe cells in vivo. Undifferentialed cells may also increase the possibility of a malignancy or olher tumor fonning at the site of the therapeutic implant, or by migration of transplanted cells.
This invention is directed towards a strategy in which undifferentiated cells remaining in such differentiated cell populations can be depleted. This is effected by genetically aitering the cells, so that a gène that is lethal to a cell In which It Is expressed, or renders It susceptible to a lethal effect of an extemal agent, Is placed under transcriptlonal control of a genetic élément that causes II to be expressed preferentiaily in any undifferentiated cells in the population. This is a négative sélection strategy, designed to minimize the proportion of undifferentialed celfs. It ts possible to combine this technique with positive sélection techniques of various kinds, in order to obtain reiatively pure populations of the desired tissue type that are essentially free of undifferentiated cells.
As a non-limiling vaJidation of the Invention, human embryonic stem (hES) celis hâve been transduced with an adenovirus veclor (TPAC) in which a herpès virus thymidine kinase gène was placed under control of a promoter séquence for human telomerase reverse transcriptase (hTERT). hES cells constitutively express hTERT, but this ability is lost upon differentiation. Example 10 (Figures 6-8) show that transduction of hES cells wilh TPAC veclor renders undifferentialed cells susceptible to lethalily by the prodrug ganciclovir, a substrate for thymidine kinase, at a concentration of - 20 µM. Example 11 (Figure 9) shows that when hES cells are transduced with TPAC vector and then differentiated with DMSO, ihere are no surviving cells with détectable 0CT*4 expression (a phenotipe of undifferentiated cells).

The techniques of this invention are designed in part to provide cell populations with improved characlerislics for human therapy. After depleting undifferenliated cells, the differentiated population is expected to possess better functional and engraftment characteristics, and hâve reduced risk of creating unwanted lissue architecture and malignancies in the treated subject. in addition, cell populations depleted of undifferentiated cells are more homogeneous, which provides a distinct advantage for non-therapeutic applications, such as producing antibody, cDNA libraries, and screening drug candidates.
Définitions
Prototype "primate Plurîpotent Stem celis" (pPS cells) are pluripotent cells derived from pre-embryonic, embryonic, or fetal lissue at any time after fertilization, and hâve the characteristic of being capable under appropriale conditions of producing progeny of several différent cell types Ihat are derivatives of ail of the îhree germinal layers (endoderm, mesoderm, and ectoderm), according to a standard art-accepled test, such as the ability to fomi a leratoma in 8-12 week old SCID mice.
Included in the définition of pPS cells are embryonic cells of various types, exemplified by human embryonic stem (hES) cells, described by Thomson et al. (Sdence 282:1145, 1998); embryonic stem cells from other primates, such as Rhésus stem cells (TTiomson et al., Proc. Nall. Acad. Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et a!., Biol. Reprod. 65:254, 1996) and human embryonic germ (hEG) cells (Shamblott et ai., Proc. Natl. Acad. Sci. USA 95:13726,1998). Olher types of pluripotent cells are also included In the term. Any cells of primate origin thaï are capable of producing progeny that are derivatives of ail three genninal layers are included, regardiess of whether they were derived from embryonic lissue, fetal tissue, or other sources. This invention relates to pPS cells that are not derived from a malignant source. It is désirable (but not atways necessary) that the ceils be kan/otypically normal.
pPS cell cultures are described as "undifferentiatecT v^^hen a substantlal proportion of stem cells and thelr derivatives in the population display morphological characteristics of undifferentiated cells, clearly distinguishing them from differentiated cells of embryo or adult origin. Undifferentiated pPS cells are easily recognized by those skilled In the art, and typlcally appear In ihe tv;o dimensions of a microscopic view in colonies of cells with high nudear/cytoplasmic ratios and prominent nucieoli. Il is understood that colonies of undifferentiated cells within the population v^ill oflen be surrounded by neighboring cells that are differentiated. Nevertheless» the undifferenliated colonies persist v^hen the population is cultured or passaged under appropriate conditions, and individual undifferentiated cells constitute a substantlal proportion of the cell population. Cultures that are subsfantîally undifferenliated contain al least 20% undifferentiated pPS ceds, and may contain at least 40%, 60%, or 80% in order of Increasing préférence. Whenever a culture or cell population is refen-ed lo in this disclosure as proliferating *Svithout differentiation". what is meant Is that after prolifération, the composition Is subslantialiy undifferenliated according to the precedlng définition.
"Feeder cells" or 'leeders" are terms used to describe cells of one type that are co-cullured with cells of anolher type, to provide an environment in which the cells of the second type can grow. The feeder cells are optionally from a différent species as the cells they are supporting. For example, certain types of pPS cells can be supported by primary mouse emb^onic fibroblasts, immortalized mouse embryonic fibroblasts, or human flbroblast-like cells differentiated from hES cells, as described later in this disclosure. pPS cell populations are said io be "essentially free" of feeder cells if the cells hâve been grown Ihrough at least one round after spiitting in which fresh feeder cells are not added to support the grov/th of the pPS. Cultures essentially free of feeder cells contain iess than about 5% feeder cells. Whenever a culture or cell population is referred to in this

disclosure as leeder-free", what is meant is that the composition is essentially free of feeder cells according to the preceding définition, subject only to further constralnts expiicitîy requlred.
The term "embryoid faodies" is a term of art synonymous with "aggregate bodles". The terma refer to aggregales of dlfferentiated and undifîerentiated cells that appear when pPS ceils overgrow In monolayer cultures, or are maintained in suspension cultures. Embryoid bodies are a mixture of différent cell types, typically from several germ layers, distlngulshable by morphologicai crileria.
The terms "commilted precursor cells", "llneage restricled precursor cells" and "restricted deveiopmentai Hneage cells" ail reîer to cells that are capable oî proliîerating and differentiating into several différent cell types, with a range that is typically more limited than pluripotenl stem cells of embryonic origin capable of giving rise to progeny of ail three germ layers. Non-limiting examples of committed precursor cells include hematopoietic cells, which are pluripotent for various blood cells; hepatocyte progenitors, which are pluripolent for bile duct epithelial cells and hepalocytes; and mesenchymal stem cells. Another example is neurai restricted cells, v;hich can générale glia) cell precursora Ihat progress to oligodendrocytes and astrocytes, and neuronal precursors that progress to neurons.
For the purposes of thîs description, the term "stem cell" can refer to elther a pluripotent stem cell, or a committed precursor cell, both as defined above. Minlmally, a stem cell has the ability to proliferate and form ceils of more than one différent phenotype, and is alao capable of self renewai — either as part of the same culture, or when cultured under différent conditions. Embryonic slem cells can be identified as positive for the enzyme lelomerase.
As used in this disclosure, "differenliated" and "undifferentiated" are relative terms depending on the context in which they are used. Specifically, in référence to a particular type of self-renewing stem cell, the temi "undifferentiatecT refers back to the same self-renewing stem cell, whereas the term "differentiatecT refers to one or more of the relatively mature phenotypes Ihe stem ceil can générale — as discernable by morphologicai criteria. anligenic markers, and gène transcripts they produce. Undifferentiated pPS cells hâve the ability to differentiate into ail three genn layers. The cells differentlated from them do not. and can readily be recognized by one skilled in the art by morphologicai criteria.
The terms "polynucleolide" and "nucleic acid molécule" refer to a polymer of nucleotîdes of any length. Included are gènes and gène fragments, mRNA, tRNA, rRNA, ribozymes, cDf^JA, recombinant polynucleotides, branched polynucleotides, plasmkis, vectors, isolated DNA and RNA, nucleic acid probes, and primers. As used in this disclosure, the term polynucleotides refer interchangeably to double- and single-stranded molécules. Uniess otherwise specified or required, any embodiment of the Invention that is a polynucleotide encompasses bolh a double-stranded form, and each of the two complementary single-stranded fonms known or predkrted to make up the double-stranded form. Included are nucleic acid anaîogs such as phosporamidates and thiophosporamidates.
A cell is said to be "genetically altered", "transfected", or "genellcally transformed" when a polynucleotide has been transferred into the cell by any suitable means of artlflclal manipulation, or where the cell is a progeny of the origfnally aftered cell that has fnherited the polynucleotide. TTie polynucîeoiide will often comprise a transcribable séquence encoding a protein of interest, which enables the cell to express the protein at an elevated level. The genetic altération is said to be "inheritable" if progeny of the altered cell hâve the same altération.
A "control elemenf or "control séquence" is a nucleotide séquence involved In an interaction of molécules that contribuîes to the functional régulation of a polynucleotide, such as replication, duplication, transcriplion, splicing, translation, or dégradation of the polynucleolide. Transcriptional control éléments include promoters, enhancers. and repressors.

Particular gene sequences referred to as promoters, like Ihe 'TERT promoter", or the "OCT-A promoter", are poiynucleotide sequences derived from the gene referred to that promote transcription of an operatively iinked gene expression product. It is recognized that various portions of the upstream and inlron untranslated gene sequence may in some instances contribule to promoter activity, and that ail or any subset of these portions may be present in the genetically engineered construct referred to. The promoter may be based on the gene sequence of any species having the gene, unless expllcilly restricted, and may incorporate any additions, substitutions or deletions desirable, as long as the abilily to promole transcription in the target lissue. Genetic conslructs desîgned for trealment of humans lypically comprise a segment thaï is at least 90% identicaf to a promoter sequence oï a human gene. A particular sequence can be tested for activity and specificity, for example, by operatively linking to a reporter gene (Example 9).
Genetic elements are said to be "operatively Iinked" if they are in a structural relationship permitting them to operate in a manner according to their expected function. For instance, if a promoter helps initiate transcription of the coding sequence, the coding sequence can be referred to as operatively Iinked to (or under contre! of) the promoter. There may be intervenîng sequence between the promoter and coding region so long as this functional relationship is maintained.
In the context of encoding sequences, promoters, and other genetic elements, the term "heterologous" indîcates that the element is derived from a genotypîcally distinct entrty from that of the rest of the enllty to which II Is belng compared. For example, a promoter or gene introduced by genetic engineering techniques inlo an animal of a different species is said to be a heteroîogous poiynucleotide. An "endogenous" genetic element is an element that is in the same place in the chromosome where it occurs in nature, although other elements may be artificially introduced into a neighboring position.
The terms "polypeptide", "peptide" and "protein" are used interchangeably in this disclosure to refer to polymsrs of amino acids of any length. The polymer may comprise modified amino acids, it may be linear or branched, and it may be interrupted by non-amino acids.
General Techniques
For further elaboration of gêneral techniques useful in the practice of this invention, the practitioner can refer to standard textbooks and reviews in ced biology, tissue culture, and embryology. Included are Teratocarclnomas and embryonic stem cells: A practicat approach (E.J. Robertson, ed., IRL Press Ud. 1987); Guide to Techniques in Mouse Deve/opment (P.M. Wasserman et a!., eds„ Academie Press 1993); Embryonic Stem Cell Diffeœntiation in Wro (M.V. Wiles, Melh. Enzymoi. 225:900, 1993); Properiies and uses of Embryonic Stem CeUs: Prospects for AppHcation to Human Biology and Gene Therapy (P.D. Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998). Differentiation of stem cells is reviev/ed in Robertson, Meth. Cell Biol. 75:173,1997; and Pedersen. Reprod. Fertil. Dev. 10:31,1998.
Methods In molecular genelics and genetic engineering are described generally în the current editions of Motecu/ar Cioning: A Laboralory Manuai, (Sambrook et al.); OJigonucieottde Synîhesis (M.J. Gait, ed.,); Ar)imai Cell Culture (R.l. Freshney, ed.); Gene Transfer Vectors for Mammafian Cells (Miller & Calos, eds.); Currenf Protocols in Molecular Biology and Shori Protocols in Molecular Biology, 3rd Edition (F.M. Ausubel et al., eds.); and Recombinant DNA Methodology [B. Wu ed., Academie Press). Reagents, cioning vectcrs, and kits for genetic manipulation referred to in this disclosure are available from commercial vendcrs such as BtoRad, Stratagene, Invitrogen, and ClonTech.
General techniques in cell culture and media collection are outlined in Large Scale Mammallan Cell Culture (Hu et al„ Curr. Opln. Biotechnol. 8:148. 1S97); Scrum-free Media (K. Kitano, Biotechnology 17:73,

1991); Large Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2:375,1991); and Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19:251,1990). Other observations about the media and their impact on the culture environment hâve been made by Marshall McLuhan and Fred Allen.
Sources of Stem Cells
This Invention can be pracliced using stem celis of various types, which may include the following non-limîling examples.
U.S. Patent 5,851,832 reports multipotent neural stem cells obtained from brain tissue. U.S. Patent 5,766,948 reports producing neuroblasts from newborn cerebral hemispheres. U.S. Patent 5,654,183 and 5,849,553 report the use of mammalian neural crest stem cefis. U.S. Patent 6,040,180 reports In vitro generation of differentiated neurons from cultures of mammalian mullipotential CNS stem cells. WO 98/50526 and WO 99/01159 report generation and isolation of neuroepitheliai stem celis, oligodendrocyte-astrocyte precursors, and lineage-restricted neuronal precursors. U.S. Patent 5,968,829 reports neural stem celis obtained from embryonic forebrain and cultured with a medium comprising glucose, transferrin, insulin, selenium, progesterone, and several other growth faclors.
Primary Iiver cell cultures can be obtained from human biopsy or surgically excised tissue by perfusion with an appropriate combination of coliagenase and hyaluronidase. Allernativeiy. EP 0 953 633 Al reports isolaling liver cells by preparing minced human liver tissue, resuspending concentrated tissue celis in a growth medium and expanding the cells in culture. The growth medium comprises glucose, insulin, transferrin, T3, FCS, and various tissue extracts that ailow the hepatocytes to grow without malignanl transformation. The cells in the liver are thought to contain specialized cells Includlng liver parenchymal cells, Kupffer cells, sinusoïdal endolhelium, and bile duel epithelium, and aIso precursor cells {referred to as "hepatoblasts" or^oval cells*) that bave the capacity to differenliate inlo both mature hepatocytes or biliary epithelial cells (LE. Rogler, Am. J. Pathol. 150:591, 1997; M. Alison, Current Opin. Cell Biol. 10:710, 1998; Lazaro et al.. Cancer Res. 58:514. 1998).
U.S. Patent 5,192,553 reports methods for isolating human neonatal or telal hematopoiettc stem or progenitor cells. U.S. Patent 5,716,827 reports human hematopoietic celis that are Thy-1 positive progenitors, and appropriate growth media to regenerate them in vitro, U.S. Patent 5,635.387 reports a method and device for culturing human hematopoietic cells and their precursors. U.S. Patent 6,015.554 describes a method of reconstituting human lymphoid and dendritic cells.
U.S. Patent 5,486,359 reports homogeneous populations of human mesenchymal stem cells that can differentiate into cells of more than one connective tissue type, such as bone, cartilage, tendon, ligament, and dermts. They are obtained from bone marrow or periosteum. AIso reported are culture conditions used to expand mesenchymal stem cells. WO 99/01145 reports human mesenchymal stem cells isolated from perlpheral blood of Individuels treated with growth factors such as G-CSF or GM-CSF. WO 00/53795 reports adipose-derived stem cells and laltices, substantially frea of adipocytes and red celis. These cells reportedly can be expanded and cultured to produc© hormones and conditioned culture media.
The invention can be practiced using stem celis 0I any vertebrale species. Included are slem cells from humans; as well as non-human primates, domestic animais, livestock, and other non-human mammals.
Amongst the stem cells suitable for use in Ihis invention are primate pluripotent stem (pPS) cells derived from tissue formed after gestation, such as a blastocyst, or îetal or embryonic tissue taken any time during gestation. Non-limiting examples are primary cultures or eslablîshed Unes of €mbf>'onic stem cefls.

Media and Feeder Cells
Media for isolating and propagating pPS cells can hâve any of several different formulas, as long as the cells obtatned have the desired characteristics, and can be propagated further. Suitable sources are as foliows: Dulbecco's modified Eagles medium (DMEM), Gibco # 11965-092; Knockout Dulbecco's modified Eagles medium (KO DMEM), Gibco # 10629-018; 200 mM L-glutamine, Gibco # 15039-027; non-essentlal amino acid solution, Gibco 11140-050; p-mercaptoethanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco # 13256-029. Exemplary serum-containing ES medium is made with 80% DMEM (lypically KO DMEM), 20% defined fetal bovine serum (F3S) nol heat inactivated, 0.1 mM non-essential amino acids, 1 mM L-glutamine, and 0.1 mM g-mercaptoethanol. The medium is filtered and stored at 4*C for no longer ïhan 2 weeks. Seaim-free ES medium is made with 80% KO DMEM, 20% serum replacement, 0.1 mM non-essential amino acids, 1 mM L-glutamîne, and 0.1 mM p-mercaptoethanol. An effective serum replacement is Gibco # 10828-028. The medium is filtered and stored at 4'C for no longer than 2 weeks. Just before use, human bFGF is added io a final concentration of 4 ng/mL (Bodnar et al., Geron Corp, International Patent Publication WO 99/20741).
Feeder cells (where used) are propagated In mEF medium, containing 90% DMEM (Gibco # 11965-092). 10% FBS (Hyclone # 30071-03), and 2 mM glutamine. mEFs are propagated in T150 flasks (Corning # 430825), splitting the cells 12 every other day with trypsin, keeping the cells subconfluent. To prepare the feeder celi layer, celis are irradiated at a dose to inhibit proliferation but permit synthesis cf important factors that support hES cells (-4000 rads gamma in-adiation). Six-well culture plates (such as Falcon # 304) are coated by incubation at 37o'C with 1 mL 0.5% geiatin per well overnight, and piated with 375.000 irradiated mEFs per well. Feeder celi layers are typically used 5 h to 4 days after plating. The medium is replaced with fresh hES medium just before seeding pPS cells.
Conditions for culturing other stem cells are known, and can be optlmized appropriately according to the cell type. Media and cuilure techniques for pariicular cell types referred to in the previous section are provided in Ihe references cited.
Embryonic Stem Cells
Embryonic stem cells can be isolated from blastocysts of members of the primate species (Thomson et al.. Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells can be prepared from human blastocyst celis using the techniques described by Thomson et al. (U.S. Patent 5,843,780; Science 282:1145.1998; Curr. Top. Dev. Biol. 38:133 ff.. 1998) and Reubinoff et ai. Nature Blotech. 18:399,2000.
Briefly, human blastocysts are obtalned from human In vivo preimplantation embryos. Altematlvely, In vitro fertilized (IVF) embryos can be used, or one celi human embryos can be expanded to the blastocyst stage (Bongso et al.. Hum Reprod 4: 706,1989). Human embryos are cultured lo the blastocyst stage in G1.2 and G2.2 medium (Gardner et al., Fertil. Slerii. 69:84, 1998). Blastocysts that develop are selected for ES cell isolation. The zona pellucida is removed from blastocysts by brief exposure to pronase (Sigma). The înner cell masses are isolated by Immunosurgery, in which blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antisenjm for 30 minutes, then washed for 5 minutes three times in DMEM, and exposed to a 1:5 dilution of Guinea pig complement (Gibco) for 3 minutes (see Solter et al., Proc. Natl, Acad. Sci. USA 72:5099, 1975). After two further washes in DMEM, iysed trophectoderm cel!s are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM piated on mEF feeder layers.
After 9 to 15 days, inner cell mass-derived outgrowths are dissociated inîo clumps either by exposure to calcium and magneslum-free phosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispase or trypsin, or by mechanical dissociation with a micropipelte; and then repiated on mEF in fresh medium.

Dissocialed cells are replated on mEF feeder layers in fresh ES medium, and observed for colony formation. Colonies demonstraling undifferentiated morphology are individually selected by micropipelte, mechantcally dissocialed into clumps, and replated. ES-like morphology is characlerized as compact colonies with apparentiy high nucleus to cytoplasm ratio and prominent nucleoli. Resulling ES celis are then routinely split every 1-2 vveeks by brief tri'psinization, exposure to Dulbecco's PBS (without calcium or magnesium and with 2 mM EDTA), exposure to typa iV collagenase (-200 U/mL; Gibco) or by selection of individual colonies by micropipelte. Clump sizes of about 50 to 100 cells are optimal.
Embryonic Germ Cells
Human Embryonic Germ (hEG) cells can be prepared from primordial germ celis present In human fetal material taken about 8-11 weeks after the last menslrual period, Suitable preparation methods are described in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726,1998 and U.S. Patent 6,090,622.
Briefly, genital ridges are rinsed with Isotonic buffer, Ihen placed into 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution (BRL) and cut into Nlnety-six well tissue culture plates are prepared with a sub-confluent layer of feeder cells cultured for 3 days in modified EG grcwth medium tree of LIF. bFGF or forskolin, inactivated with 5000 rad -irradiation. Suitable feeders are STO cells (ATCC Accession No. CRL1503). -0.2 mL of primary genn cell (PGC) suspension is added to each of ihe v;ell3. The first passage is conducted after 7-10 days in EG growth medium, transferrlng each well to one well of a 24-well culture dlsh prevlously prepared with irradialed STO mouse fibrobiasts. The cells are cultured with daiiy replacement of medium untii cell morphology consistent with EG cells are observed, typicaliy after 7-30 days or 1-4 passages.
Propagation of pPS Cells in an Undifferentiated State
pPS cells can be propagated continuously in culture, using a combination of culture conditions thaï promote proliferation without promoting differentiation.
Traditionally, pPS cells are cultured on a layer of feeder cells, typicaliy fibroblast type cells, often derived from embryonic or fetal tissue. The cell lines are plated to near confluence, usualiy irradiated to prevenl proliferation, and then used to support pPS cell cultures.
in one illustration, pPS cells are first derived and supported on primary embryonic fibrobiasts. Mouse embryonic fibrobiasts (mEF) can be ofatained from outbred CF1 mfce (SASCO) or other suitable slrains. The abdomen of a mouse at 13 days of pregnancy is swabbed with 70% ethanol, and the decidua is removed into phosphate buffered saline (PBS). Embryos are harvested; placenta, membranes, and soft lissues are removed; and the carcasses are washed twice in PBS. They are ihen transferred to fresh 10 cm bacterial dishes containing 2 mL trypsin/EDTA, and ftnely minced. After incubating 5 min af 37oC , the trypsin is

inacllvaled with 5 mL DMEM containing 10% bovind sérum (FBS), and the mixture is transferred to a 15 mL conicaf tube and dissociated. Débris is allowed to setfle for 2 min, the supernatant is made up to a final volume of 10 mL, and piated onto a 10 cm lissue culture plate or T75 flask. The flask is incubated undisturbed for 24 h,


of 100-1000 cells in the nnixed population after -11 days. After brief collagenase treatment, the îibroblast-like cells can be collecled under a microscope, passaged in mEF medium, and tested for their ability to condition ES medium.
Medium that has been conditioned for 1-2 days is typically used to support pPS cell culture for 1-2 days, and then exchanged. If deslred, conditioned medium can be supplemenîed before use with addilional grovnh factors that benefit pPS cell culture. For hES, a gro;vth facîor like bFGF or FGF-4 can be used. For hEG, culture medium may be supplemented with a growth factor like bFGF, an inducer of gp130, such as LIF or Oncostatin-M, and perhaps a factor that elevâtes cyclic AMP levels, such as forskolin.
Characteristics of Undifferentiated pPS Cells
\n the two dimensions of a standard micrcscopic image, hES cells hâve high nuclear/cytoplasmic ratios in the plane of the image, prominent nucleoli, and compact colony formation with poorly discernable cell junctions. Cell lines can be karyotyped using a standard G-banding technique (available at many clinical diagnostics labs that provides routine karyotyping services, such as the Cytogenetics Lab at Oakiand GA) and compared to publîshed human karyotypes.
hES and hEG cells can a!so be characterized by expressed cell markers. In gêneral, the tissue-specific markers discussed in this disclosure can be detected using a suitable Immunological technique — such as flow cytometry for membrane-bound markers, immunohislochemistry for intracellular markers, and enzyme-linked Immunoassay, for markers secreted Into the medium. The expression of prolein markers can aiso be detected at the mRNA level by reverse Iranscriptase-PCR using marker-specific primera. See U.S. Patent 5.843,780 for further details.
Slage-specinc embryonic antigens (SSEA) are characteristic of certain embryonic cell types. Antibodies for SSEA markers are available from the Developmentai Studies Hybridoma Bank (Bethesda MD). Other useful markers are detectable using antibodies designated Tra-1-50 and Tra-1-81 (Andrews et aL, Cell Unes from Human Germ Cell Tumors, in EJ. Robertson, 1987, supra). hES cells are typically SSEA-1 negative and SSEA-4 positive, hEG cells are typically SSEA-1 positive. Diflerentlation of pPS cells In vitro results in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression and Increased expression of SSEA-1. pPS cells can aiso be characterized by the presence of alkallne phosphatase activity, which can be detected by fixing the cells with 4% paraformaldehyde, and then developing with Vector Red as a substrate, as described by the manufacturer (Vector l-aboratories, Burlingame CA).
Embryonic stem cells are aiso typicaDy telomerase positive and OCT-4 positive. Telomerase activity can be determined using TRAP activity assay (Kim et al., Science 266:2011, 1997), using a commercially available kit (TRAPeze® XK Telomerase Detection Kit, Cat. s7707; intergen Co., Purchase NY; or TeloTAGGGTM Telomerase PCR EUSApIus, Cat. 2,013.89; Roche Diagnostics, Indianapolis). hTERT expression can aiso be evaiuated at the mRNA level by RT-PCR. The LightCycier Telo TyAGGG™ hTERT quantification kit (Cat. 3,012,344; Roche Diagnostics) is available commercially for research purposes.
Dlfferentiation pPS Cells
Differentiation of the pPS can be initiated by first forming embryoid bodies. General principles in culturing embryoid bodies are reported in O'Shea, Anat. Rec. (New Anat. 257:323, 1S39). pPS cells are cullured in a manner that permits aggregates to form, for which many options are available: for exampîe, by overgrowth of a donor pPS cell culture, or by culturing pPS celts In culture vessels having a substrale with low adhesion properties which allows EB formation. Embryoid bodies can ailso be made In suspension culture.

pPS ceils are harvested by brief collagenase digestion, dissociated into clusters, and plated in non-adherent cell culture plates. The aggregales are fed every few days, and then harvested after a suitable period, typically 4-8 days. The ceils can then be cuitured m a médium and/or on a substraîe ihat promûtes enrichment of celis of a particular lineage. The substrate can comprise matrix compcnents such as Matrigel® (Becton Dickenson), laminin, collagen, gelatin, or matrix prcduced by first culturing a matrlx-producing cell Une (such as a fibroblast or endothelial cell Une), and then lysing and v;ashing in such a v/ay that the matrix remains attached to the surface of the vessel. Embryoid bodies comprise a helerogeneous cell population, potentially having an endoderm exterlor, and a mesoderm and ectodemi interior.

General principals for obtaining tissue ceils from pluripotent stem ceils are reviewed in Pedersen (Reprod. Fertil. Dev. 6:543. 1994), and U.S. Patent 6,090,622. Other publications of interest include the following: For neural progenitors, neural restrictive ceils and glial cell precursors, see Bain et al., Biochem. Biophys. Res. Commun. 200:1252. 1994; Trojanowski et al., Exp. Neurol. 144:92, 1997; Wojdk et al., Proc. NaO. Acad. Sel. USA 90:1305-130; and U.S. Patents 5,851.832, 5,928.947, 5,766,948, and 5.849.553. For cardiac muscle and cardiomyocytes see Chen et a!., Dev. Dynamics 197:217, 1993 and Wobus et al., Differenliation 48:173, 1991. For hematopoletic progenitors, see Burkert et al., New Biol. 3:698, 1991 and Biesecker et al., Exp. Hematol. 21:774, 1993. U.S. Patent 5,773.255 relates to glucose-responsive insulin secreting pancreatic beta cell lines. U.S. Patent 5,789,246 relates to hepatocyte precursor cells. Other progenitors of interest include but are not limiled to chondrocytes, osteoblasts, reîinal pigment epithelial ceils, flbroblasts, skin cells such as keratinocytes, dendritic celis, hair follicle cells, rénal duct epithelial cells, smooth and skeletal muscle cells, testicular progenitors, and vascular endothelial cells.
Scienlists at Geron Corporation hâve discovered that culturing pPS cells or embryoid body cells in the présence of ligands that bind growth factor receptors promotes enrichment for neural precursor cells. The growth environmenl may contain a neural cell supportive extraceilular matrix, such as fibronectin. Suitable


Markers independent of HNF-4a expression Include α-anlitrypsin, αfeîoproîein, apoE, glucokinase, insulin growth factors 1 and 2, lGF-1 receptor, insulin receptor, and leptin. Markers dépendent on HNF-4α expression


A suitable microarray analysis is conducted using a Genetic Microsystems array generator, and an Axon GenePixTM Scanner. Microarrays are prepared by amplifying cDN'A fragments in a 96 or 384 well fonnat, and then spotted direclly onto glass slides. To compare mRNA prépara tions îrom two cell populations, one préparation is converled into Cy3-labeled cDNA, while the other is converled into Gy5-iabeled cDNA. The two cDNA préparations are hybridized simultaneousiy to the microarray sitde, and then v;ashed to eiiminate non-

spécifie binding. Any given spot on the array will bind each cf the cDNA products in proportion to abundance of the transcript in tha two original mRNA préparations. The siide is then scanned at wavelengths appropriate for each of the labels, and the relative abundance of mRNA is determined. Preferabïy, the ievel of expression oi the effector gène will be at least 5-foid or even 25-fold higher in the undifferentiated cells relative to the differentiated cells.


Immunol. Immunother. 42:115, 1996; Rodriguez et al., Prostate 34:259, 1998; Mauceri et al., Cancer Res. 56:4311; 1996.
AIso suitable are genes that tnduce or mediate apoptosis — such as the ICE-family of cysteine proteases, the Bcl-2 family of proteins, Bax, bclXs and caspases (Favrot et al., Gene Ther. 5:728,1998; McGill et al.. Front. Biosci. 2:D353, 1997; McDonnell et al., Semin. Cancer Biol. 6:53, 1995). Another potential antl-lumor agent is apoptin, a protein that induces apoptosis even where small drug chemotherapeutics fail (Pletersen et al., Adv. Exp. Med BIol. 465:153, 2000). Koga et al. (Hu. Gene Ther. 11:1397, 2000) propose a lelomerase-specific gene therapy using the hTERT gene promoter llnked to the apoptosis gene Caspase-8 (FLtCÊ). Gu et al. (Cancer Res. 60:5359, 2000) reported a binary adenoviral System that induced Bax expression via the hTERT promoter. They found that it elicited tumor-specific apoptosis in vitro and suppressed tumor growth in nude mice.
AIso of interest are enzymes present in the lytic package that cytotoxic T lymphocytes or LAK cells deliver to their targets. Perforin, a pore-forming protein, and Fas ligand are major cytolytic molecules in these cells (Brandau et al., Clin. Cancer Res. 6:3729, 2000;Cruz et al., Br. J. Cancer 81:881,.1999). CTLs also express a family of at least 11 serine proteases termed granzymes, which hâve four primary substrate specificities (Kam et al., Biochim. Biophys. Acta 1477:307, 2000). Low concentrations of streplolysin Gand pneumolysln facllitale granzyme B-dependent apoptosis (Browne et al., Mol. Cell Biol. 19:8604,1999).
Other suitable effectors encode polypeptides having activity that is not itself toxic to a cell, but renders the cell sensrtive to an otherwise nontoxic compcund — either by melabolicatly allering the oeil, or by changing a non-loxic prodrug into a lethal drug. Le,thality to progeny with an undifferenlialed phenotype only ocours when the prodnjg is present Thus, the prodrug can be combined with the cells while they are being differentiated, expanded, or maintained in vitro, to minimiza the proportion of cells with undifferentiated phenotype. The reader wiil readity appreciate that the prodnjg can also be given to a patient being treated with the cells, either simultaneousty with the treatment, or at a subsequent time, in order to minimize the emergence of progeny v;ith an undifferentiated phenotype in vivo.
Exemplary effector genes v.ith this property encode thymldine kinose {tk), such as may be derived from a herpes simptex virus, and calalytlcaliy equivalent variants. The HSV tk converts the anli-herpelic agent ' ganciclovlr (GCV) to a toxic product that interferes with DNA replicalion in proliferating cells.
U.S. Patent 5,631,235 (Baylor College of Medicine) outlines adenoviral vectors containing an HSV îk gene operatively llnked to a promoter that expresses tk in cancer cells. U.S. Patent 5,997.859 and EP 702084 B1 (Chircn) pertain to replicalion-defective recombinani retrovirus, carrying a vector construcl directing expression oï HSV tk gene for converting an othenivise inert compound into a cytotoxic form. EP415731 AI, EP 657540 AI, and EP 657541 AI {Wellcome Foundation) propose retroviral vectors encoding an enzyme such as VZV tk, carboxypeptidase G2, alkaline phosphatase, penicillin-V amidase, and cytosine deaminase, for converting a prodnjg into an agent toxic to a cancer cell. Intemational Patent Publications WO 98/14593 and WO 00/46355 (Geron Corporation) describe constructs comprising HSV tk under control of hTERT promoter sequences.
The human HSV tk--gene sequence is provided below (SEQ. 10 N0S:2 & 3), along with illustrations of its use to targel cells expressing TERT. Simultaneously or fcllowing expression of the gene in target cells, a convertible prodrug such as ganciclovir is added lo the environmenl to effect depletion of the targets.
Another type of effector that renders the cell susceptible to an otherwise non-toxic agent is a gene that causes presentation of a foreign antigen on the cel! membrane. The presented substance may be an alloantigen, a xenoanligen, or an antigen from a non-mammalian species for which specifie antibody is readily available. Expression of the gene leads to presentation of the antigen on undifferentiated celis, which then can











supplemented with hypoxamhine and thymidine (HAT médium). Residual undifferentiated cells can be removed from the population by incubation with HAT medium. Transcripts of essential gènes such as HGPRT and TK can also be targeted using other types ci effector séquences — for example, antisense

polynucleotides, ribozymes, or encoding sequences for dominant negative analogs that lack a functional catalytic domain.
The effector region used in the vectors of this invention can be constructed so as lo be functionally controlled by a molecular switch. Fusions between the ligand-binding domain of receptors resuit in molecules in which the normal function of the native proteln is inhibiled in the absence of the hormone recognized b/ the ligand-binding domain. A fusion protein la constructed comprising a switch molecule binding domain coupled to an effector domain, in such a way that binding of a ligand (such as a small molecule hapten) to the binding domain unmasks or activâtes the effector domain. Suitable ligand binding domains can be taken from a receptor (such the estrogen receptor) or an anlibcdy. The effector domain can be any of the proleins already listed that are iethal to the cell, such as peptide toxins, endonucteases (meganucleases such as 1-Sce -1 cr humanized versions of standard restriction endonucleases), or mediators of apoptosis. The Iethal function of the effeclor gene is quiescent. uniess the ligand is present. This provides another System where the effector gene renders the ce)) susceptible to toxic effecîs of an externa) agent (in this case the ligand), which can be administered at will to control the depletion of undifferentîated cells, either in culture or in vivo.
The vector constructs for use in this invention can also conlain a positive selection marker, such as an antiblotic resistance gene, that is also under control of the specific promoter. Exemplary is a vector having the configuration hTERT promoter - tk gene - IRES - neo. This is designed so that both the suicide effector and drug resistance gene are expressed under control of the TERT promoter. The intemal ribosome entry site (1RES) sequence allows bolh the tk gene and the neo gene to be under Iranscriptional control of the hTERT promoter. A post-lranslational cieavage site, such as 2A sequences (Felipe et al., Gene Ther. 6:1 SB. 1999( can be used to similar effect. Generation and selection of hES Unes that hâve stably integraîed such a construct is facilitated by the activily of the drug resistance gene in the undifferentiated celîs. This has an advantage over co-transfection methods using a drug resistant gene under control of a different prom.oter, because the drug resistance gene will net be expressed in differentïated ceils. This should avoid an unwanted immunological response by the hcsî against the gene product in transplanted cells.
Selection Techniques to Elliinate Undifferentiated Celis
To deplete differentïated cell populations of undifferentiated cells, the effector gêne is selectively expressed In the undifferentiated cells.
This can be accomplished in severai ways. In one embodiment, the population is geneticaily altered using a vector in which a transcriptional control element of ths appropriate specificity is operatively Ilnked to the effector gene. The genetic alteration may be transient (for example, using an adenovirus vector), meaning that the level of expression diminishes as the ceils divide. This is suitable for generating differentiated cell populations that will be free of heterologous genes at the tfme of therapy. The genetic alteration may also be permanent (for example, using a retrovlral vector), meaning that the alteration is inherltable by progeny of the iniîially altered cell. This is suitable for generating differentiated celi populations that will hâve an ongoing correclive function as they proliferate In vitro or In vivo, to elîmînate any undifferentiated ordedifferentiated celis that arise in the population.
Any suitable expression vector can be used. Suitable viral vector Systems for producing stem ceils altered according to this invention can be prepared using commerciaiîy available virus components. Virai vectors comprising effector genes are generally descrited in the publications referenced in the last section. Alternatively, vector plasmids can be intrcduced into ceils by electroporation, or using lipid/DNA complexes, such as those described in U.S. Patent Nos. 5,578,475; 5,627,175; 5,705.308; 5,744.335; 5.976.557; 6,020,202; and 6,051,429. Exemplary is the formulation Lipofectamine 2000TM, available from Gibco/Liîe


bromodeoxyuridine (to sélect for TK négative celis). Ahematîvely, the transfecîion could be done with just the RNAi kit, in which case geneticin is cmilîed from the medium, After isolation of the surviving clones, thèse lines are induced to differentiate to the desired cell type, and exposed lo HAT médium to kill any residual stem cells. Celi populations may be obtained using thèse techniques that are "depleted" o( undifferentiated cells, which indlcates any significant réduction in the proportion of undifferentiated ce!!s présent. After the procédure









Cells were washed, stained with DAPl and mounted. The staining was typicailly performed -2 days after passaging. Cells were a!so examined for expression of alkaline phosphatasa, a marker for undifferentiated ES cells. This was performed by culluring the cells on chamber slides, fixing with 4 % paraformaldehyde tor 15







was added to the welIs to reach a final volume of 3.5 mL and the mixture was left on the cells overnight. In other experiments the DNA/Iipid mixture was added to vvefis containing sufficient mEF-conditioned médium such that the final volume was 3.5 mL and the cells were incubated in this mixture overnight.













Figure 6 shows the results of this experimenl. No change in total cell number was detected at MOI ot 30 in the abssnce of GCV; but there was some decrease at MOI of 100 in absence of GCV starling at 48 h. Evidence for toxîcity of GCV alone was detected: wells receiving GCV alone contained approximately 55% as

many cells as the control wells on day 2. diminishing to 40% by day 4. Wells receiving GRN376 at MOls of 30 or 100 cultured In the présence of GCV showed identical results: by day 2, thèse wells contained 18% of the cells contained in the conttol wells, while at days 3 and 4 thèse wells contained 6% and 8% of the cells in the control wells.






Figure 11 (boltom) shows sensitivily of the TPAC lines to a différent prodrug, (E)-5-(2-bromoviny1)-2'-deoxyuridine (BVDU). Although a significant decrease on cell counls was observed with ail BVDU concentrations tested, ES cell killing was not actually observed. The number of cells présent even at high concentrations of BVDU is not reduced to the levél of the blank contrcl.




SEQUENCE DATA

I t will be recognized thaï the compositions and procedures provided in the description
can be effectively modified by those skilled in the art without departing from
the spirit of the invention embodied in the daims that follow.



CLAIMS
What Is claimed as the invention is:
1. A population of cells differentiated from primate pluripotent stem (pPS) cells cultured ex vivo, which is essentially free of undifferentiated cells.
2. The cell population of claim 1, wherein the cells contain a nucleic acid molecule comprising the structure P-X, wherein:
X is a nucleic acid sequence encoding a product that is lethal to a cell in which it is expressed, or renders a cell in which it is expressed susceptible to a lethal effect of an external agent; and
P is a transcriptional control element that causes X to be preferentially expressed In undifferentiated cells.
3. A primate pluripotenl stem (pPS) cell containing a nucleic acid molecule comprising the structure P-X,
wherein:
X is a nucleic acid sequence that is lethal to a cell in which it is expressed, or renders a cell in which it is expressed susceptible to a lethal effect of an external agent; and
P is a transcriptional control element that causes X to be preferentially expressed In undifferentiated cells.
4. The cell or cell population of claim 2 or 3, wherein X encodes a toxin, or a protein that induces or mediates apoptosis.
5. The cell or ceil population of claim 2 or 3, wherein X encodes an enzyme that converts a prodrug to a compound that is lethal to a cell in which X is expressed.
6. The cell or cell population of claim 5, wherein X encodes a thymidine kinase.
7. The cell or cell population of claims 2-6, wherein P-X is an introduced heterologous molecule.
8. The cell or cell population of claims 2-6, wherein P is an endogenous transcriptional control element.
9. The cell or cell population of claims 2-6, wherein P is an OCT-4 promoter, or a promoter of telomerase reverse transcriptase (TERT).
10. The cell or cell population of any preceding claim, wherein the stem cell(s) are human embryonic stem (hES) cells.

11. A method of producing a population of differentiated ceils, comprising
a) providing a cell population comprising undifferentiated stem cells that contain a nucleic acid molecule comprising the structure P-X, wherein X Is a nucleic acid sequence encoding a product that is lethal to a cell in which It is expressed, or renders a cell in which it is expressed susceptible to a lethal effect of an external agent, and P is a transcriptional control element that causes X to be preferentially expressed In undifferentiated ceils; and
b) causing at least some undifferentiated cells in the population to differentiate.

12. The method of claim 11, further comprising combining the cell population with the external agent.
13. A method of depleting a cell population of undifferentiated stem cells, comprising genetically altering undifferentiated stem cells in the population so that they contain a nucleic acid molecule comprising the structure P-X, wherein X is a nucleic acid sequence encoding a product that is lethal to a cell In which it is expressed, and P is a transcriptional control element that causes X to be preferentially expressed in undifferentiated cells.
14. The method of claims 11-13, wherein X encodes a toxin, or a protein that induces or mediates apoptosis.
15. A method of depleting a cell population of undifferentiated stem cells, comprising:

a) genetically altering undifferentiated stem cells in the population so that they contain a nucleic acid molecule comprising the structure P-X, wherein X renders a ceil in which It is expressed susceptible to a lethal effect of an external agent, and P Is a transcriptional control element that causes X to be preferentially expressed in undifferentiated cells; and
b) depleting undifferentiated cells from the population by combining the cells with the external agent.

16. The method of claim 11, 12 or 15, wherein X encodes an enzyme that converts a prodrug to a compound that is lethal to a cell in which X is expressed.
17. The method of claim 15, wherein X encodes a thymidine kinase.
18. The method of claim 16 or l 7, wherein the extemal agent is ganciclovir.
19. The method of claims 11-18, wherein P-X is an introduced heterologous molecule.
20. The method of claims 11-18, wherein P is an endogenous transcriptional control element.
21. The method of claims 11-20, wherein the ceil population is genetically altered such that X is transiently expressed in undifferentiated cells in the population.
22. The method of claims 11-20, wherein P-X Is Inherited by progeny of cells In the population, becoming expressed in undifferentiated progeny.

23. The method of claims 11-22, wherein P Is an OCT-4 promoter, or a promoter of telomerase reverse transcriptase (TERT).
24. The method of claims 11-23, wherein the nucleic acid molecule contains the structure P-X-Y, wherein Y is a drug resistance gene.
25. The method of claims 11-24, wherein the stem cells are human embryonic stem (hES) cells.
26. A population of differentiated cells obtained according to the method of any of claims 11-25.
27. The cell or cell population according to any of claims 1-10 or claim 25, which is a population of neuronal Cells, astrocytes, oligodendrocytes, hepalocytes, cardiomyocytes, osteoblasts, or their committed precursor cells.
28. A pharmaceutical preparation for human or animal surgery or therapy, comprising the cell or ceil population according to any of claims 1 -10 or 26-27.
29. Use of a prodrug in the preparation of a medicament for depleting undifferentiated stem cells in a human or animal body,
wherein the undifferentiated stem cells comprise the structure P-X, where:
X is a nucleic acid sequence encoding a product that converts the prodrug to a compound that is
lethal to the undifferentiated stem cells; and
P is a transcriptional control element that causes X to be preferentially expressed in
undifferentiated cells.
30. The use according to claim 29, wherein the prodrug is ganciclovir.

31. A pharmaceutical preparation for human or animal surgery r therapy substantially as herein described with reference to the accompanying drawings.
Dated this 21 day of May 2003


Documents:

0782-chenp-2003 abstract-duplicate.pdf

0782-chenp-2003 claims-duplicate.pdf

0782-chenp-2003 description (complete)-duplicate.pdf

0782-chenp-2003 drawings-duplicate.pdf

782-chenp-2003 power of attorney.pdf

782-chenp-2003-claims.pdf

782-chenp-2003-correspondnece-others.pdf

782-chenp-2003-correspondnece-po.pdf

782-chenp-2003-description(complete).pdf

782-chenp-2003-drawings.pdf

782-chenp-2003-form 1.pdf

782-chenp-2003-form 13.pdf

782-chenp-2003-form 3.pdf

782-chenp-2003-pct.pdf


Patent Number 229151
Indian Patent Application Number 782/CHENP/2003
PG Journal Number 12/2009
Publication Date 20-Mar-2009
Grant Date 13-Feb-2009
Date of Filing 21-May-2003
Name of Patentee GERON CORPORATION
Applicant Address 230 CONSTITUTION DRIVE, MENLO PARK, CA 94025,
Inventors:
# Inventor's Name Inventor's Address
1 GOLD, JOSEPH, DANIEL 100 LUNDY'S LANE, SAN FRANSCISCO, CA 94110,
2 LEBROWSKI, JANE, STEPHANIE 150 GABARDA WAY, PORTOLA VALLEY, CA 94028,
PCT International Classification Number C12N15/00
PCT International Application Number PCT/US2001/44309
PCT International Filing date 2001-11-26
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/253,357 2000-11-27 U.S.A.
2 09/783,203 2001-02-13 U.S.A.
3 60/253,443 2000-11-27 U.S.A.