United States Patent (191
`Salser et al.
`
`[II]
`
`(45]
`
`4,396,601
`Aug. 2, 1983
`
`(54] GENE TRANSFER IN INTACT MAMMAlS
`Inventors: Winston A. Salser; Martin J. Cline,
`(75]
`both of Pacific Palisades; Howard D.
`Stang, Van Nuys, all of Calif.
`(73] Assignee: The Regents of the University of
`Calif., Berkeley, Calif.
`[21] Appl. No.: 134,234
`[22] Filed:·
`Mar. 26, 1980
`[51]
`Int. Cl.l ...................... A61K 37/48; A61K 35/14
`[52} u.s. Cl. ········································ 424/94; 424/95;
`424/101; 424/251;435/172;435/241
`(58] Field of Search ........................... 424/94, 95, 101;
`435/241, 172, 68
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`4,172,124 1011979 Koprowski et al .•.•.•••..•.....• 435/172
`
`OTHER PUBLICATIONS
`Cline et al.-Nature, vol. 284, Apr. 3, 1980, pp. 422-425.
`Hilts-The Washington Post, Oct. 16, 1980, p. A7.
`
`Orlova et al.-Chem. Abst., vol. 92 (1980), p. 20,448e.
`McElwain et ai.-Chem. Abst., vol. 92 (1980), p. 15767d.
`
`Primary Examinei'-Sam Rosen
`Attorney, Agent, or Firm-Bertram I. Rowland
`
`ABSTRACT
`(57]
`Methods and compositions are provided for gene trans(cid:173)
`fer to intact mammals with expression of the exogenous
`genetic material in the host. Mammalian host ceUs
`which are regenerative, normally highly proliferative
`or subject to induced proliferation, are transformed or
`modified in vitro with DNA capable of replication and
`expression in the host cell, wherein the DNA becomes
`incorporated into the cell. The modified cells are found
`to regenerate in the host with expression of the intro(cid:173)
`duced DNA. Particularly, mammalian cells were modi(cid:173)
`fied with genes providing for overproduction of a par(cid:173)
`ticular enzyme. The modified cells were reintroduced in
`the host under conditions providing for selective advan(cid:173)
`tage of the modified cells.
`
`15 Claims, No Drawings
`
`Sanofi/Regeneron Ex. 1 038, pg 990
`
`Merck Ex. 1038, pg 1016
`
`

`
`1
`
`4,396,601
`
`GENE TRANSFER IN INTACf MAMMALS
`
`15
`
`BACKGROUND OF THE INVENTION
`I. Field of the Invention
`The discovery that one could introduce exogenous
`genes into a bacterial host in vitro and observe expres(cid:173)
`sion of the exogenous genes in the bacterial host opened
`up vistas of new capabilities for the production of a
`wide range of compounds, particularly proteins, im- 10
`proved methods of treating waste, novel types of fertil(cid:173)
`izers, and new vaccines. While transformation of proka(cid:173)
`ryotes offer many new and yet envisaged opportunities,
`there is also great interest in being able to modify euka-
`ryotes and particularly mammalian cells.
`Many diseases are genetically related involving ge(cid:173)
`netic deficiencies, which are usually either failure to
`produce a gene product or production of an abnormal
`product. Other situations involve treatment of a host
`with drugs which may have substantial toxicity to host 20
`cells. In these instances, it would be desirable to provide
`the host with the missing capability, the normal capabil(cid:173)
`ity or a defense mechanism against the detrimental ef(cid:173)
`fects of the drug. The capability to modify a host's
`genetic structure to provide for either additional ge- 25
`netic capabilities or reparation of a defective capability
`on a temporary or permanent basis opens up wide ave(cid:173)
`nues in the treatment of genetic deficiencies and disease.
`2. Description of the Prior Art
`Methods of introducing genetic material into a host 30
`cell include viral vectors Munyon et a!. J. Virol,
`7:813-820, 1971; cell-cell fusion, the fusion to cells of a
`limited number of chromosomes enveloped in nuclear
`membranes, Fournier et a!. Proc. Nat!. Acad. Sci.
`74:319-323, 1977; and cellular endocytosis of micro- 35
`precipitates of calcium-DNA complex, Bachetti and
`Graham,
`ibid. 74:1590-1594, 1977; Maitland and
`McDougall, Cell 11:233-241, 1977; Pellicer ,eta!. ibid.
`14:133-141, 1978 and Wig1er et a!. ibid. 14:725-731,
`1978. Cell lines lacking thymidine kinase are readily 40
`transformed by appropriate DNA to a tk + status when
`grown in the presence of a folic acid inhibitor and thy(cid:173)
`midine. Pellicer, supra and Wigler, supra.
`
`2
`By use of this approach, animals were obtaind in which
`the majority of the type of cells involved contained the
`added genetic material in a functionally active state.
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`In accordance with the subject invention, a host is
`genetically modified by removing from the host or
`syngeneic source cells capable of regeneration when
`present in the host. The cells are then combined with
`DNA having genes capable of expression to provide a
`selective advantage for cells, under conditions where
`cells incorporate the DNA. The cells, which will in(cid:173)
`clude cells having the additional DNA, are then re(cid:173)
`turned to the host. The genes providing the selective
`advantage can be combined with other genetic material
`which will be incorporated in conjunction with the
`gene supplying the selective advantage. The gene pro(cid:173)
`viding the selective advantage will be referred to as the
`selective marker.
`Various methods may be employed for introduction
`of the genetic material, each of the methods having
`advantages and disadvantages. After introduction of the
`treated cells into the host, conditions are maintained in
`the host naturally, by administration of a physiologi(cid:173)
`cally active compound, or by dietary exclusion, to pro(cid:173)
`vide a selective advantage for the cells which have been
`genetically modified. In this way, genetic functions can
`be provided for a variety of purposes including treat(cid:173)
`ment of genetic deficiencies, which includes providing a
`genetic capability which the host lacks or production of
`a normal product where the host produces an abnormal
`one; production of enzymes which can protect the host
`from cytotoxic agents; or for production of a wide vari(cid:173)
`ety of proteins e.g. hormones, globulins or the like.
`In describing the invention, the host and host cells
`will be considered first, followed by the genetic mate(cid:173)
`rial which may be employed for modifying the host
`cells and the manner in which the host cells are modi(cid:173)
`fied, and concluding with the regeneration of the modi(cid:173)
`fied cells and the purposes and effect of expression of
`the genetic material introduced into the modified cells.
`
`45
`
`SUMMARY OF THE INVENTION
`Methods and compositions are provided for provid(cid:173)
`ing mammalian hosts with additional genetic capability,
`either a novel capability or enhancement of an existing
`one. Host cells capable of regeneration are removed and
`treated with genetic material under conditions whereby 50
`the genetic material is introduced into the host cells and
`becomes capable of replication and expression. The
`introduced genetic material includes at least one marker
`which allows for selective advantage for the host cells
`in which the introduced genetic material is capable of 55
`expression. The host cells are returned to the host under
`regenerative conditions, preferably of rapid prolifera(cid:173)
`tion of the cells, optionally with stressing of the host to
`provide a selective advantage for the genetically modi(cid:173)
`fied cells. It is found under these conditions, that the 60
`modified cells proliferate and express the genetic mate(cid:173)
`rial which was introduced. Particularly, genetic mate(cid:173)
`rial was employed which provided for expression of an
`enzyme. Either under the normal conditions of the host
`or subjecting the host to an enzyme antagonist, a selec- 65
`tive proliferative advantage for the modified cells hav(cid:173)
`ing overproduction of the enzyme resulted, in contrast
`to the normal cells incapable of such overproduction.
`
`, Host and Host Cells
`Various mammalian hosts may be treated in accor(cid:173)
`dance with the subject invention, such as homo sapiens
`and domestic animals, particularly bovine, equine,
`ovine and porcine. The type of host cell which will be
`employed is one which is capable of regeneration, pref(cid:173)
`erably rapid proliferation, either naturally or induced;
`can be isolated from the host or syngeneic source; can
`be modified by introduction of genetic material, which
`genetic material will then be capable of expression and
`replication; can be maintained in vitro, so as to be re(cid:173)
`turned to the host in a viable state; are capable of being
`returned to the source in the host; and can provide the
`added genetic function in a form which is useful to the
`host.
`Among potential cells which may be employed are
`bone marrow cells, particularly stem cells which pro(cid:173)
`vide hematopoietic functions. Other examples of tissues
`which have persistent stem cells included the intestinal
`mucosa and the germ line tissues. Use of these tech(cid:173)
`niques to introduce genes into germ line cells may be of
`especial interest in breeding improved strains of domes(cid:173)
`tic animals. Other cells which can be employed include
`cells of regenerative organs e.g. liver. Any body mem-
`
`Sanofi/Regeneron Ex. 1038, pg 991
`
`Merck Ex. 1038, pg 1017
`
`

`
`4,396,601
`
`3
`ber which is regenerative or can be induced to regener(cid:173)
`ate can be a source of cells.
`Bone marrow cells chosen for modification should
`optimally be populations rich in stem cells. Further(cid:173)
`more, the cells chosen are preferably dividing, rather
`than stationary cells. To increase the fraction of these
`types of cells, the host may be treated by various tech(cid:173)
`niques to increase the level of proliferating cells. For
`example, vinca alkaloids may be employed which in(cid:173)
`hibit mitosis, followed by rapid proliferation of the 10
`cells.
`·
`A wide variety of genetic material (DNA) may be
`employed to provide for the selective marker. The se(cid:173)
`lective marker will allow for rapid proliferation of the
`modified cells in the host under normal conditions of 15
`the host or where rapid proliferation is subject to inhibi(cid:173)
`tion. The inhibition can be as a result of introduction of
`a drug which inhibits (a) proliferation because of inter(cid:173)
`ference with transcription of DNA or translation of
`RNA, that is, expression of one or more genes; (b) cell 20
`membrane formation; (c) cell wall formation, (d) en(cid:173)
`zyme activity; or (e) combination thereof.
`A wide variety of drugs are known which are em(cid:173)
`ployed for the treatment of disease which inhibit cell
`replication, so as to favor the host against a parasitic 25
`invader such as bacteria, protozoa, or even a neoplastic
`variant of the host cell. The effectiveness of the drug
`may be inhibited in a cell by introducing into the cell
`genes which express an enzyme which reacts with the
`drug to deactivate it, genes which overproduce an en- 30
`zyme involved in the metabolic pathway which the
`drug inhibits, so as to provide a selective advantage for
`the cells having higher concentrations of the enzyme(s),
`or genes which would provide for a metabolic pathway
`less affected by the drug, than the endogenous meta- 35
`bolic pathway.
`Alternatively, the enzyme can provide for increased
`production of a metabolite essential to mitosis e.g. a
`metabolite on the biosynthetic pathway to DNA or
`RNA, for example, the formation of nucleosides. The 40
`modified cells having the selective marker which pro(cid:173)
`vides for enhanced enzyme production permits the
`modified cells to compete more effectively for a limited
`amount of metabolite precusor against the wild type
`cell.
`The genetic material which is employed for recombi(cid:173)
`nation with the host cells may be either naturally occur(cid:173)
`ring, synthetic, or combinations thereof. Depending
`upon the mode employed for introduction, the size of
`the genetic material introduced will vary. Furthermore, 50
`when two or more genes are to be introduced they may
`be carried on a single chain, a plurality of chains, or
`combinations thereof. Restrictions as to the size of a
`DNA fragment will be as a result of limitations due to
`the technical aspects of the vector: if a recombinant 55
`DNA is to be used, by the packaging requirements of a
`viral vector; the probability of transfer into the recipient
`cells by the method employed; the manner of prepara(cid:173)
`tion and isolation of the DNA fragments; or the like.
`The selective markers employed can be chosen to 60
`deactivate an antimetabolite to mammalian cells, by
`reacting with the antimetabolite and modifying the
`antimetabolite to an ineffective product. Various en(cid:173)
`zymes and their genes are known and have been isolated
`for deactivating drugs. The most numerous examples 65
`are bacterial enzymes which deactivate antibiotics, such
`as those enzymes which confer resistance to amino(cid:173)
`glycosides and polymyxines (streptomycin, kanamycin,
`
`45
`
`4
`neomycin, amikacin, gentamicin, tobramycin, etc.), and
`the like. Another drug which may find use is PALA.
`Where the drug does not provide a selective advantage,
`since the host metabolic pathways are not involved, a
`gene providing resistance to such a drug would not be
`useful. Illustrative of this situation are sulfonamides,
`which block a bacterial pathway, but not a mammalian
`metabolic pathway.
`Alternatively, rather than providing a gene which
`expresses an enzyme, one could provide a gene which is
`not subject to interference by the drug. For example,
`one could employ DNA having a mutation at the site at
`which the drug binds or DNA which results in RNA or
`a protein, which substantially reduces the binding of the
`drug to the site at which the drug is active. Illustrative
`of drugs which are active by binding to specific sites are
`the macrolides, e.g. erythromycin and aminoglycosides,
`e.g. streptomycin.
`The next group of drugs are chemotherapeutic
`agents. Protection of the host cells from the chemother(cid:173)
`apeutic agents may be provided by introducing genes
`which overproduce the enzyme inhibited by the drug or
`deactivate the drug. Illustrative drugs include metho(cid:173)
`trexate, which inhibits dihydrofolate reductase, purine
`analogs, which interfere with the enzymes involved
`with inosinic acid, and pyrimidine analogs, such as fluo(cid:173)
`rouracil, which inhibits thymidine monophosphate syn(cid:173)
`thesis.
`The selective marker may provide for enhanced pro(cid:173)
`duction of one or more metabolites involved in prolifer(cid:173)
`ation, for example, production of nucleotides or nucleo(cid:173)
`sides. An illustrative gene is the gene which codes for
`thymidine kinase, which is involved in the biosynthetic
`pathway to thymidylic acid. This selective advantage
`need not be associated with antimetabolite administra(cid:173)
`tion to the host.
`In some genetic diseases the gene which corrects the
`genetic defect may itself confer a replicative advantage.
`For example, the insertion of genes for adenosine deam(cid:173)
`inase into cells of the marrow of certain patients with
`combined immunodeficiency disease may confer a se(cid:173)
`lective advantage upon the replication of their stem
`cells leading to the production of a large population of
`immunocompetent cells which will ameliorate the ef(cid:173)
`fects of the disease.
`Finally, one may employ genes which provide for
`production of a protein other than an enzyme, which
`allows for selective advantage of the modified cells. For
`example, this can be as a result of production of inducer
`which prevents repression of translation to provide
`semiconstitutive or constitutive production of an en(cid:173)
`zyme. In such cases a regulator gene may confer selec(cid:173)
`tive advantage even when no drug is employed.
`In summation, the types of DNA which will be em(cid:173)
`ployed for selective markers include genes which react
`with drugs which interfere with regeneration so as to
`destroy activity of the drug; genes which provide sites
`which arc not susceptible to drug action, so as to pre(cid:173)
`vent the drug's action in the particular cell; genes which
`are repetitive for production of a desired protein e.g. an
`enzyme, which is inhibited by the drug; or genes which
`affect the regulatory function of the cell, so as to pro(cid:173)
`vide for overproduction of a particular enzyme by the
`natural processes of the cell, and which increase the
`normal replication of the cell genes to enable the cell to
`better compete for limited resources within the body.
`If a drug is to be employed for providing the selective
`advantage the gene employed must be appropriately
`
`Sanofi/Regeneron Ex. 1 038, pg 992
`
`Merck Ex. 1038, pg 1018
`
`

`
`4,396,601
`
`6
`a wild type gene for correct expression of a protein.
`With bone marrow stem cells, genes could be provided
`with
`the correct
`sequence
`to correct hemo-
`globinopathes, such as sickle cell disease and thalasse(cid:173)
`mia. Other defects could include defects in the produc(cid:173)
`tion of'plasma coagulation factors, e.g. fibrinogen, pro-
`thrombin and the various Factors, especially Factors
`VIII and IX. By introducing genes providing for struc(cid:173)
`turally normal proteins fulfilling these functions, in
`conjunction with the ability to provide selective pres(cid:173)
`sures for the modified cells, the modified cells may be
`maintained in the host of a high level for extended peri(cid:173)
`ods of time.
`Depending upon the nature of the cells, the cells may
`be introduced into the host in various ways. With bone
`marrow or liver cells, the cells may be introduced intra(cid:173)
`venously. It may be desirable to treat the host to reduce
`the relevant cell population so that rapid cell replication
`will be favored. Various techniques can be employed to
`achieve this result, such as the use of mitotic inhibitors,
`e.g. vinca alkaloids, irradiation with X-rays, or other
`technique. It is desirable that prior to the introduction
`of the modified cells to the host, the host have a low
`level of the relevant cell type so that after introduction,
`25 there may be a rapid and expanding proliferation of the
`modified cells.
`After introduction of the modified cells into the host,
`the host will be stressed with relevant drug(s) if these
`are to be employed to provide selective pressure for the
`modified cells. Appropriate levels of the drug may be
`maintained to insure proliferation of the desired cells.
`Depending upon the drug, the nature of the cells, and
`~he concerns with repetitive introduction of modified
`host cells, the drug treatment may be of relatively short
`or long term duration. It is found that even after termi(cid:173)
`nation of the treatment with the drug providing the
`selective pressure, the cells continue to proliferate and
`may be maintained at a high level for extended periods
`of time.
`The following examples are offered by way of illus(cid:173)
`tration and not by way of limitation.
`
`5
`related to the drug. The particular drugs employed
`must be considered as to level of toxicity and effect on
`the particular tissue which is being modified. Also to be
`considered is the purpose of the modification, which
`may limit the involved drug. In other cases the appro- 5
`priate selective marker may be related to correction of
`the genetic deficiency involved with the disease or may
`alter the cells proliferation in any of various ways.
`A number of wa:rs have been developed for insertion
`of genetic materials into cells. Included among these 10
`techniques are viral vectors, Munyon et a!., supra; cell(cid:173)
`cell fusion involving the fusion' to cells of a limited
`number of chromosomes enveloped in nuclear mem(cid:173)
`branes, Fournier and Ruddle, supra; cellular endocyto-
`sis of microprecipitates of calcium-DNA complex, Ba- 15
`chetti and Graham, supra, Maitland and McDougall,
`supra, Pellicer et al., supra and Wigler et a!., supra:
`minicell fusion; fusion with liposomes containing DNA;
`fusion with bacterial protoplasts containing plasmid
`DNA; and fusion with erythrocyte ghosts packaged 20
`with DNA. Each of the techniques has advantages and
`disadvantages, such as efficiency of information inser(cid:173)
`tion, selectivity as to the particular nature or informa(cid:173)
`tion of the DNA, permissible size of the DNA fragment,
`and the like.
`When employing the microprecipitates of calcium(cid:173)
`DNA complex, the DNA employed may provide for a
`single gene, a single set of genes, e.g. the beta-globin
`gene cluster, or a plurality of unrelated genes. As previ(cid:173)
`ously indicated, the size of the DNA fragments will 30
`vary, depending upon the particular manner used to
`introduce the genetic information. The mixtures of
`DNA. which are not covalently linked may be intro(cid:173)
`duced by congression, that is, different fragments of
`DNA will frequently concurrently enter a suspectible 35
`cell, so that those cells which have the selective marker
`are also likely to have the genetic capability of the addi(cid:173)
`tional genes.
`The presence of a selective marker allows for selec(cid:173)
`tive pressure for preferential regeneration of the modi- 40
`fied cell. Thus, in situations where gene deficiencies
`exist which would not provide for selective advantage
`of a modified cell, the selective marker affords this
`capability. With bone marrow cells, the cells could be
`modified by introducing genes which would provided 45
`for the correction of genetic deficiencies, by expression
`of products in which the host is deficient or provide for
`
`EXPERIMENTAL
`The following is a flow chart of the progress of the
`experimentation:
`
`FIG.l
`
`Day -3
`
`Day 0
`
`Day 77
`
`Pretreat
`marrow~
`
`Mix T6T6 and Ca
`Isolate
`1:1 and inject
`marrow
`from~ cells into~
`X-rayed CBA/Ca
`donor mice:
`mice
`
`Treat with Mtx
`at intervals
`
`~
`
`Sanofi/Regeneron Ex. 1 038, pg 993
`
`Merck Ex. 1038, pg 1019
`
`

`
`8
`
`Day -3
`donors
`with
`VLB
`
`7
`
`-continued
`DayO
`
`CBtCa
`
`Mock trans·
`formation
`with "wild
`type" DNA
`
`CBAf6T6
`
`Transform
`with
`MtxRDNA
`
`4,396,601
`
`Day 77
`(1) Analyze karyotype
`(2) Transfer marrow
`to secondary
`irradiated CBA/Ca
`
`mi<;et
`
`Mt•r•tment
`
`Analyze karyotypes
`DHFR levels
`Hematologic smtus
`
`Transformation of Mouse Bone Marrow In Vitro
`Mouse fibroblast Swiss 3T6 cells highly resistant to 20
`Mtx and containing reiterated structural genes specify(cid:173)
`ing DHFR were employed (See Kellems et a!. J. Bioi.
`Chern. 254, 309-318, 1979). They were maintained in
`4 X J0-4 M methotrexate (Mtx) and designated 3T6 Rl.
`DNA was isolated from 3T6 Rl and from non-resistant 25
`(wild type) mouse cell lines including 3T6 (fibroblastic)
`and Ll210 (lymphocyctic leukemia) and in later experi(cid:173)
`ments from salmon sperm (Sigma). The relative ratio of
`dihydrofolate reductase synthesis and number of gene
`copies in 3T6 Rl and 3T6 was approximately 30 to I. 30
`DNA coprecipitated with calcium phosphate was used
`to transform wild type Ll210 cells to methotrexate
`resistance by the method of Bachetti and Graman, su(cid:173)
`pra, as modified by Wigler et a!. supra.
`Equal numbers ofCBA/Ca and CBA/H-T6T6 mice 35
`were injected intraperitoneally with 3 or 4 mg/kg of the
`mitotic inhibitor vinblastine 3 days before marrow was
`removed for in vitro transformation. Mitotic inhibition
`by this treatment is followed by a burst of proliferation.
`Assays of colony-forming cells (CFU-S), when com- 40
`pared with total cell counts, showed that suspensions
`from animals thus treated were relatively depleted of
`mature cells and enriched approximately 3-fold in plu(cid:173)
`ripotent spleen colony-forming cells (CFU-S). On the
`day of transformation (designated day 0, FIG. I) single 45
`cell suspensions in McCoy's SA medium with 15% fetal
`calf serum were obtained from femurs and tibias of
`sacrificed animals.
`Cells from Ca and T6T6 animals were placed in sepa(cid:173)
`rate pools. All T6T6 animals had the chracteristic 50
`marker chromosome abnormality. Cell suspensions of
`5 X J06 in 10 ml complete medium were incubated with
`1.0 ml Ca-precipitated DNA containing a total of 40 1-Lg
`DNA as described by Wigler eta!., supra, for 4 hours at
`37" C. in 5% C02 in tissue culture flasks. For cells to be 55
`transformed to Mtx resistance, either 2 or 4 1-Lg of DNA
`was from the 3T6Rl cell line. During this period differ(cid:173)
`entiated phagocytic marrow cells firmly adhered to the
`flask.
`T6T6 cells were incubated with DNA from 3T6 Rl 60
`Mtx-resistant cells, and CBA/Ca marrow cells were
`incubated with control DNA preparations from Mtx(cid:173)
`sensitive cells. Thereafter, loosely adherent cel]s·were
`collected and centrifuged at 150Xg for 10 min and
`resuspended in DNA-free complete medium. After 65
`careful cell counts, Ca and T6T6 cells were combined in
`a ratio of 1:1 and between SX J06 and SX J07 of the
`combined cells were injected intravenously into recipi-
`
`ent CBA/Ca mice in a volume of 0.3 to 0.4 ml in Mc(cid:173)
`Coy's medium with fetal calf serum. These recipients
`had received 850 rads irradiation from a cobalt source
`24 hours previously to eradicate endogenous hemato(cid:173)
`poiesis. This dose or irradiation was selected because it
`had low lethality but virtually eradicated endogenous
`spleen colonly-forming cells (CFU-S). Thus an average
`of 2± I endogenous CFU-S after 850 rads and 0.5±0.5
`endogenous CFU-S after 900 rad whole body irradia(cid:173)
`tion was observed in this mouse strain. Between 48 and
`96 hours after injection, the recipient animals began
`treatment with the previously established Mtx protocol.
`
`Hematopoietic Effects of Methotrexate Treatment in
`the Mouse
`An appropriate schedule of Mtx treatment which
`would select for drug-resistant hematopoietic cells
`without lethality in control animals was established as
`follows. Groups of normal CBA or C3H mice weighing
`between 18 and 25 g were treated by a thrice weekly
`schedule of intraperitoneal injections of Mtx in doses
`varying between 0.5 and 8 mg/kg per injection. An
`escalating schedule of 0.5 mg/kg for 4 doses, 2 mg/kg
`for 4 doses and then 4 mg!kg thrice weekly was se(cid:173)
`lected as not lethal but having profound suppressive
`effects on hematopoiesis. Tibial cellularity, peripheral
`white cell counts and hematocrits were all depressed in
`Mtx-treated animals and megaloblastic morphologic
`changes developed in the bone marrows. The hemato(cid:173)
`crit and tibial cellularity were found to be the easiest
`and most reliable hematologic parameter to follow and
`remained depressed in animals continuously treated
`with Mtx for at least 3 months. False elevations of he(cid:173)
`matocrit in Mtx-treated mice were occasionally ob(cid:173)
`served in sick and dehydrated animals. No difference in
`sensitivity to Mtx was observed in the mouse strains
`CBA/Ca and CBA/H T6T6 as measured by standard
`hematologic parameters over 3 months of observation.
`
`Selection of Drug Resistance Marrow Cells
`The irradiated mice receiving mixtures of control Ca
`cells _and T6T6 cells transformed with 3T6 Rl DNA
`were treated with Mtx for periods of 24 to 77 days. At
`intervals, animals were sacrificed or subjected to a limb
`amputation to obtain bone marrow samples. These were
`analyzed for karyotype distribution, celiularity, CFU-S
`content and injected into secondary irradiated CBA/Ca
`recipients. The results of two initial experiments are
`shown in Tables I and II.
`
`Sanofi/Regeneron Ex. 1 038, pg 994
`
`Merck Ex. 1038, pg 1020
`
`

`
`9
`TABLE I
`EXPERIMENT MB2. KARYOTYPE ANALYSIS bF
`MARROW CELLS OF IRRADIATED CBA/Ca MICE
`RECEIVING. A 1:1 MIXTURE OF CONTROL Ca and
`TRANSFORMED T6T6 MARROW CELLS
`Duration of Mtx Treatment
`Karyotype
`(days)
`(% T6T6)
`57
`59
`79
`67
`97
`93
`84
`
`Recipient•
`
`Primary 1
`Primary 2
`~econdary 2
`Primary 3
`Secondary 3a
`Secondary 3 b
`Secondary 3c
`
`0-24
`0-32
`32-46
`0-39
`39-53
`39-67
`39-73
`
`4,396,601
`
`10
`• .TABLE III -continued
`KARYOTYPE ANALYSIS OF BONE MARROW AND
`PLURIPOTENT STEM CELLS FROM CBA/Ca MICE ·
`RECEIVING 1:1 MIXTURE OF CONTROL Ca AND'
`TRANSFORMED T6T6 BONE MARROW CELLS
`Bone Marrow
`Karyotype of
`Karyotype
`SEleen Colonies
`T6T6
`T6T6
`Ca Mixed
`(%)
`(%)
`(%)
`(%)
`
`Duration or
`Recipient Mtx (days)
`
`5
`
`10
`
`Primary 2
`Primary 3
`
`0-40
`0-47
`
`75
`74
`
`57
`58
`
`26
`8
`
`17
`33
`
`•trradioted CBA/Ca recipients of the 1:1 mixture of a Ca transformed with wild
`type DNA and T6T6 cells transformed wlth JT6R DNA are designated "primary" ·15
`and each mouse is given a unique number. The day of infusion is designated "0 ...
`Recipients of marrow from "primary" animals are designated "secondary·~ ond bear
`the same identifying number. Karyotype analysis of recipient bone marrow cells
`were perfonned aOer the designated interval of methotrexate treatment. Between 50
`and 100 chromosome spreads were analyzed.
`
`20
`
`25
`
`30
`
`Recipient•
`
`48-68
`
`TABLE II
`EXPERIMENT TV4. KARYOTYPE ANALYSIS OF
`MARROW CELLS OF CDA/Ca MICE RECEIVING A 1:1
`MIXTURE OF CONTROL Ca and TRANSFORMED T6
`MARROW CELLS
`Days with Mtx
`Days Without
`(%T6)
`Karyotype
`Mtx
`79
`0-33
`Primary 1
`75
`0-40
`Primary 2
`74
`0-47
`Primary 3
`83
`0-47
`Primary 3
`88, 88, too•
`47-61
`Secondary 3
`75
`0-54
`Primary 4
`83
`54-72
`Secondary 4
`Primary 5
`96
`0-65
`Primary 5
`63
`66-113
`0-65
`•Irradiated recipients of the 1:1 mixture of Ca cells transformed wlth wild t)'pe 35
`DNA and T6T6 cells transformed with 3T6Rl DNA are designated "primary'' and
`each mouse is given a unique number. The day of infusion is designated "0."
`Recipients of marrow from "primary" animals are designated ''secondary" and bear
`the same identifying number.
`• •Three secondary recipients.
`
`Individual spleen colonies were removed 10 days
`after innoculation of irradiated recipient with bone
`marrow cells. A single cell suspension was made
`from each colony and cells were incubated with
`colcemide 3 f.Lglml for 90 minutes before treatment
`with hypotonic KCL and fixation with acetic acid(cid:173)
`/ethanol for chromosome spreads.
`In order to analyze whether the predominance of
`T6T6-marked cells involved pluripotent stem cells as
`well as other proliferating marrow cells, marrow was
`taken from selected primary recipient animals and
`5 X 104 cells were injected into irradiated recipient
`CBA/Ca mice in a typical spleen colony-forming
`(CFU-S) assay. (Tell and McCulloch Rad. Res. 14:213,
`1961) Ten days later the secondary recipients were
`killed and individual spleen colonies removed for
`karyotype analysis. As seen in Table III the percentage
`of T6T6 karyotype predominated in the pluripotent
`marrow stem cell population. Mixed T6T6-Ca spleen
`colonies were also seen, presumably resulting from
`development of T6T6 colonies on a background of
`endogenous hematopoiesis in the Ca animals.
`
`Effect of Drug Administration on Cell Predominance
`In order to assess the significance of these results,
`control experiments were performed to determine
`whether T6T6-marked cells had any proliferative ad(cid:173)
`vantage or increased resistance to Mtx and to analyze
`the contribution of endogenous hematopoietic repopu(cid:173)
`lation in irradiated CBA/Ca animals. Experimental
`animals receiving an equal mixture of mock transformd
`Ca and mock transformed T6T6 and either untreated or
`treated with Mtx for up to two months had a predomi(cid:173)
`nance of Ca karyotypes liS anticipated from the contri(cid:173)
`butions of infused Ca cells and endogenous Ca cells.
`TABLE IV
`KARYOTYPE ANALYSIS OF MARROW CELLS OF
`CONTROL Ca MICE RECEIVING A 1:1 MIXTURE OF
`MOCK TRANSFORMED Ca AND MOCK TRANSFORMED
`T6T6 MARROW CELLS
`Duration of 1\-ltx Treatment
`(days)
`
`Recipient•
`
`Karyotype
`(% T6T6)
`
`Between roughly days 30 and 40 a clear increase in 40
`the percentage of bone marrow cells displaying the
`T6T6 marker· was observed in primary recipient ani(cid:173)
`mals. Marrow from these mice was·injected into irradi(cid:173)
`ated secondary recipients which were then treated with
`methotrexate. They, too, showed an increased ratio of 45
`T6T6 to Ca karyotypes, above that seen in the primary
`marrow recipients. Seven such experiments were per(cid:173)
`formed and this same pattern was seen in five indepen(cid:173)
`dent experiments involving 19 primary recipient ani(cid:173)
`mals and 30 secondary recipients. Only two experi- 50
`ments during this same period failed to show a predomi(cid:173)
`nance of transformed karyotype.
`When methotrexate treatment of animals receiving
`transformed marrow cells was stopped, the predomi(cid:173)
`nance of T6T6 karyotypes persisted for at least 3 weeks 55
`(Primary Recipient 3, Table III) but gradually dimin(cid:173)
`ished by 8 weeks without treatment (Primary Recipient
`5, Table II).
`--------------T~A~B=L=E~I=II~---