United States Patent
`
`[19]
`
`Salser et al.
`
`[1 1]
`
`[45]
`
`4,396,601
`
`Aug. 2, 1983
`
`[54] GENE TRANSFER IN INTACI‘ MAMMALS
`
`[75]
`
`Inventors: Winston A. Salser; Martin J. Cline,
`both of Pacific Palisades; Howard D.
`Stang, Van Nuys, all of Calif.
`
`[73] Assignee:
`
`The Regents of the University -of
`Calif., Berkeley. Calif.
`
`[211 App]. No.: 134,234
`
`[22] Filed:‘
`
`Mar. 26, 1980
`
`Int. C1.3 .................... .. A61K 37/48; A6lK 35/14
`[51]
`[521 US.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 10/I979 Koprowski et al.
`
`.............. .. 435/172
`
`OTHER PUBLICATIONS
`
`Cline et al.—Nature, vol. 234, 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 al.—Chem. Abst., vol. 92 (1980), p. 15767d.
`
`Primary Examiner——Sam Rosen
`Attorney, Agent, or Firm—Bertram I. Rowland
`
`[57]
`
`ABSTRACT
`
`Methods and compositions are provided for gene trans-
`fer to intact mammals with expression of the exogenous
`genetic material
`in the host. Mammalian host cells
`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-
`duced DNA. Particularly, mammalian cells were modi-
`fied with genes providing for overproduction of a par-
`ticular enzyme. The modified cells were reintroduced in
`the host under conditions providing for selective advan-
`tage of the modified cells.
`
`15 Claims, No Drawings
`
`Genzyme Ex. 1002, pg 44
`
`Genzyme Ex. 1002, pg 44
`
`

`
`1
`
`4,396,601
`
`GENE TRANSFER IN INTACT gMAMMALS
`
`BACKGROUND. OF THE INVENTION
`1. Field of the Invention
`The discovery that one could introduce exogenous
`genes into a bacterial host in vitro and observe expres-
`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-
`proved methods of treating waste, novel types of fertil-
`izers, and new vaccines. While transformation of proka-
`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-
`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
`cells. In these instances, it would be desirable to provide
`the host with the missing capability, the normal capabil-
`ity or a defense mechanism against the detrimental ef-
`fects of the drug. The capability to modify a host’s
`genetic structure to provide for either additional ge-
`netic capabilities or reparation of a defective capability
`on a temporary or permanent basis opens up wide ave-
`nues in the treatment of genetic deficiencies and disease.
`2. Description of the Prior Art
`Methods of introducing genetic material into a host
`cell
`include viral vectors Munyon et al. J. Virol,
`7:813—820, 1971; cell-cell fusion, the fusion to cells of a
`limited number of chromosomes enveloped in nuclear
`membranes, Foumier et al. Proc. Natl. Acad. Sci.
`74:3l9—323, 1977; and cellular endocytosis of micro-
`precipitates of calcium-DNA complex, Bachetti and
`Graham,
`ibid.
`74:l590—l594,
`1977; Maitland and
`McDougall, Cell 1l:233—241, 1977; Pellicer.et al. ibid.
`142133-141, 1978 and Wigler et al.
`ibid.
`l4:725—731,
`1978. Cell lines lacking thymidine kinase are readily
`transformed by appropriate DNA to a tk+ status when
`grown in the presence of a folic acid inhibitor and thy-
`midine. Pellicer, supra and Wigler, supra.
`SUMMARY OF THE INVENTION
`
`Methods and compositions are provided for provid-
`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
`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
`expression. The host cells are returned to the host under
`regenerative conditions, preferably of rapid prolifera-
`tion of the cells, optionally with stressing of the host to
`provide a selective advantage for the genetically modi-
`fied cells. It is found under these conditions, that the
`modified cells proliferate and express the genetic mate-
`rial which was introduced. Particularly, genetic mate-
`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-
`tive proliferative advantage for the modified cells hav-
`ing overproduction of the enzyme resulted, in contrast
`to the normal cells incapable of such overproduction.
`
`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
`
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`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-
`clude cells having the additional DNA, are then re-
`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-
`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-
`cally active compound, or by dietary exclusion, to pro-
`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-
`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-
`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-
`rial which may be employed for modifying the host
`cells and the manner in which the host cells are modi-
`fied, and concluding with the regeneration of the modi-
`fied cells and the purposes and effect of expression of
`the genetic material introduced into the modified cells.
`
`‘ Host and Host Cells
`Various mammalian hosts may be treated in accor-
`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-
`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-
`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.
`
`60
`
`Among potential cells which may be employed are
`bone marrow cells, particularly stem cells which pro-
`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-
`65 niques to introduce genes into germ line cells may be of
`especial interest in breeding improved strains of domes-
`tic animals. Other cells which can be employed include
`cells of regenerative organs e.g. liver. Any body mem-
`
`Genzyme Ex. 1002, pg 45
`
`Genzyme Ex. 1002, pg 45
`
`

`
`3
`bet which is regenerative or can be induced to regener-
`ate can be a source of cells.
`Bone marrow cells chosen for modification should
`optimally be populations rich in stem cells. Further-
`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-
`niques to increase the level of proliferating cells. For
`example, vinca alkaloids may be employed which in-
`hibit mitosis, followed by rapid proliferation of the
`cells.
`'
`
`A wide variety of genetic material (DNA) may be
`employed to provide for the selective marker. The se-
`lective marker will allow for rapid proliferation of the
`modified cells in the host under normal conditions of
`the host or where rapid proliferation is subject to inhibi-
`tion. The inhibition can be as a result of introduction of
`a drug which inhibits (a) proliferation because of inter-
`ference with transcription of DNA or translation of
`RNA, that is, expression of one or more genes; (b) cell
`membrane formation; (c) cell wall formation, (cl) en-
`zyme activity; or (e) combination thereof.
`A wide variety of drugs are known which are em-
`ployed for the treatment of disease which inhibit cell
`replication, so as to favor the host against a parasitic
`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-
`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-
`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
`modified cells having the selective marker which pro-
`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-
`nation with the host cells may be either naturally occur-
`ring, synthetic, or combinations thereof. Depending
`upon the mode employed for introduction, the size of
`the genetic material introduced will vary. Furthermore,
`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
`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-
`tion and isolation of the DNA fragments; or the like.
`The selective markers employed can be chosen to
`deactivate an antimetabolite to mammalian cells, by
`reacting with the antimetabolite and modifying the
`antimetabolite to an ineffective product. Various en-
`zymes and their genes are known and have been isolated
`for deactivating drugs. The most numerous examples
`are bacterial enzymes which deactivate antibiotics, such
`as those enzymes which confer resistance to amino-
`glycosides and polymyxines (streptomycin, kanamycin,
`
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`4,396,601
`
`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-
`apeutic agents may be provided by introducing genes
`which overproduce the enzyme inhibited by the drug or ’
`deactivate the drug. Illustrative drugs include metho-
`trexate, which inhibits dihydrofolate reductase, purine
`analogs, which interfere with the enzymes involved
`with inosinic acid, and pyrimidine analogs, such as fluo-
`rouracil, which inhibits thymidine monophosphate syn-
`thesis.
`
`The selective marker may provide for enhanced pro-
`duction of one or more metabolites involved in prolifer-
`ation, for example, production of nucleotides or nucleo-
`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-
`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-
`inase into cells of the marrow of certain patients with
`combined immunodeficiency disease may confer a se-
`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-
`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-
`zyme. In such cases a regulator gene may confer selec-
`tive advantage even when no drug is employed.
`In summation, the types of DNA which will be em-
`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 are not susceptible to drug action, so as to pre-
`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-
`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
`
`Genzyme Ex. 1002, pg 46
`
`Genzyme Ex. 1002, pg 46
`
`

`
`4,396,601
`
`5
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`l0
`
`15
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`20
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`25
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`30
`
`6
`5
`A
`a wild type gene for correct expression of a protein.
`related to the drug. The particular drugs employed
`must be considered as to level of toxicity and effect on .
`With bone marrow stem cells, genes could be provided ..
`with the
`correct
`sequence
`to
`correct
`hemo-
`the particular tissue which is being modified. Also to be
`globinopathes, such as sickle cell disease and thalasse-
`considered is the purpose of the modification, which
`mia. Other defects could include defects in the produc-
`may limit the involved drug. In other cases the appro-
`priate selective marker may be related to correction of
`tion of plasma coagulation factors, e.g. fibrinogen, pro-
`thrombin and the various Factors, especially Factors
`the genetic deficiency involved with the disease or may
`alter the cells proliferation in any of various ways.
`VIII and IX. By introducing genes providing for struc-
`turally normal proteins fulfilling these functions,
`in
`A number of ways have been developed for insertion
`of genetic materials into cells. Included among these
`conjunction with the ability to provide selective pres-
`sures for the modified cells, the modified cells may be
`techniques are viral vectors, Munyon et al., supra; cell-
`cell fusion involving the fusion to cells of a limited
`maintained in the host of a high level for extended peri-
`ods of time.
`number of chromosomes enveloped in nuclear'mem-
`Depending upon the nature of the cells, the cells may
`branes, Fournier and Ruddle, supra; cellular endocyto-
`be introduced into the host in various ways. With bone
`sis of microprecipitates of calcium-DNA complex, Ba-
`marrow or liver cells, the cells may be introduced intra-
`chetti and Graham, supra, Maitland and McDougall,
`venously. It may be desirable to treat the host to reduce
`supra, Pellicer et al., supra and Wigler et al., supra:
`the relevant cell population so that rapid cell replication
`minicell fusion; fusion with liposomes containing DNA;
`fusion with bacterial protoplasts containing plasmid
`will be favored. Various techniques can be employed to
`achieve this result, such as the use of mitotic inhibitors,
`DNA; and fusion with erythrocyte ghosts packaged
`with DNA. Each of the techniques has advantages and
`‘ e.g. vinca alkaloids, irradiation with X-rays, or other
`technique. It is desirable that prior to the introduction
`disadvantages, such as efficiency of information inser-
`of the modified cells to the host, the host have a low
`tion, selectivity as to the particular nature or informa-
`level of the relevant cell type so that after introduction,
`tion of the DNA, permissible size of the DNA fragment,
`and the like.
`‘
`there may be a rapid and expanding proliferation of the
`modified cells.
`When employing the microprecipitates of calcium-
`After introduction of the modified cells into the host,
`DNA complex, the DNA employed may provide for a
`the host will be stressed with relevant drug(s) if these
`single gene, a single set of genes, e.g. the beta-globin
`are to be employed to provide selective pressure for the
`gene cluster, or a plurality of unrelated genes. As previ-
`ously indicated, the size of the DNA fragments will
`modified cells. Appropriate levels of the drug may be
`maintained to insure proliferation of the desired cells.
`vary, depending upon the particular manner used to
`introduce the genetic information. The mixtures of
`Depending upon the drug, the nature of the cells, and
`the concerns with repetitive introduction of modified
`DNA. which are not covalently linked may be intro-
`duced by congression, that is, different fragments of
`host cells, the drug treatment may be of relatively short
`DNA will frequently concurrently enter a suspectible 35 or long term duration. It is found that even after termi-
`cell, so that those cells which have the selective marker
`nation of the treatment with the drug providing the
`are also likely to have the genetic capability of the addi-
`selective pressure, the cells continue to proliferate and
`tional genes.
`may be maintained at a high level for extended periods
`The presence of a selective marker allows for selec-
`of time.
`tive pressure for preferential regeneration of the modi- 40
`The following examples are offered by way of illus-
`fied cell. Thus, in situations where gene deficiencies
`tration and not by way of limitation.
`exist which would not
`rovide for selective advanta e
`- of a modified cell,
`the? selective marker affords thgis
`EXPERIMENTAL
`capability. With bone marrow cells, the cells could be
`The following is a flow chart of the progress of the
`modified by introducing genes which would provided 45 experimentation:
`for the correction of genetic deficiencies, by expression
`of products in which the host is deficient or provide for
`
`F1G 1
`'
`
`
`
`Isolate
`
`Day 0
`Mix T6T6 and Ca
`
`Day 77
`Treat with Mtx
`
`Day -3
`
`marrow
`,
`' Pretreat
`marrow —-9 from
`donor mice:
`
`hi and inject
`cells into —-9
`X-rayed CBA/Ca
`mice
`
`at intervals
`
`l
`
`Genzyme Ex. 1002, pg 47
`
`Genzyme Ex. 1002, pg 47
`
`

`
`Day -3
`donors
`
`with
`
`VLB
`
`‘ 7
`
`CBA/T6T6
`
`/I\
`
`Transform
`with
`.
`
`MtxRDNA
`
`-continued
`Day 0
`CBA/Ca
`
`/l\
`
`Mock trans-
`formation
`
`with “wild
`type” DNA
`
`4,396,601
`
`Day 77
`(1) Analyze karyotype
`
`(2) Transfer marrow
`
`V
`to secondary
`irradiated CBA/C3
`"W95
`
`Mtx treatment
`
`Analyze karyotypes
`DHFR levels
`Hematologic status
`
`'
`
`Transformation of Mouse Bone Marrow In Vitro
`
`25
`
`Cells from Ca and T6T6 animals were placed in sepa-
`rate pools. All T6T6 animals had the chracteristic 50
`marker chromosome abnormality. Cell suspensions of
`5x 106 in 10 ml complete medium were incubated with
`1.0 ml Ca-precipitated DNA containing a total of 40 pg
`DNA as described by Wigler et al., supra, for 4 hours at
`37° C. in 5% CO2 in tissue culture flasks. For cells to be
`transformed to Mtx resistance, either 2 or 4 pg of DNA
`was from the 3T6Rl cell line. During this period differ-
`entiated phagocytic marrow cells firmly adhered to the
`flask.
`T6T6 cells were incubated with DNA from 3T6 R1
`Mtx-resistant cells, and CBA/Ca marrow cells were
`incubated with control DNA preparations from Mtx-
`sensitive cells. Thereafter, loosely adherent cells‘were
`collected and centrifuged at 150><g for 10 min ‘and
`resuspended in DNA-free complete medium. After
`careful cell counts, Ca and T6T6 cells were combined in
`a ratio of 1:1 and between 5X 105 and 5x107 of the
`combined cells were injected intravenously into recipi-
`
`55
`
`’
`
`65
`
`Mouse fibroblast Swiss 3T6 cells highly resistant to 20
`Mtx and containing reiterated structural genes specify-
`ing DHFR were employed (See Kellems et al. J. Biol.
`Chem. 254, 309-318, 1979). They were maintained in
`4X10—4 M methotrexate (Mtx) and designated 3T6 R1.
`DNA was isolated from 3T6 R1 and from non-resistant
`(wild type) mouse cell lines including 3T6 (fibroblastic)
`and Ll2l0 (lymphocyctic leukemia) and in later experi-
`ments from salmon sperm (Sigma). The relative ratio of
`dihydrofolate reductase synthesis and number of gene
`copies in 3T6 R1 and 3T6 was approximately 30 to 1.
`DNA coprecipitated with calcium phosphate was used
`to transform wild type Ll210 cells to methotrexate
`resistance by the method of Bachetti and Graman, su-
`pra, as modified by Wigler et al. supra.
`Equal numbers of CBA/Ca and CBA/H-T6T6 mice
`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-
`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-
`ripotent spleen colony-forming cells (CFU-S). On the
`day of transformation (designated day 0, FIG." 1) single 45
`cell suspensions in McCoy’s 5A medium with 15% fetal
`calf serum were obtained from femurs and tibias of
`sacrificed animals.
`
`30
`
`35
`
`40
`
`ent CBA/Ca mice in a volume of 0.3 to 0.4 ml in Mc-
`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-
`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 21-1 endogenous CFU-S after 850 rads and 0.5105
`endogenous CFU-S after 900 rad whole body irradia-
`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 weretreated 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-
`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-
`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-
`matocrit in Mtx-treated mice were occasionally ob-
`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
`cellswand T6T6 cells transformed with 3T6 R1 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, cellularity, CFU-S
`content and injected into secondary irradiated CBA/Ca
`recipients. The results of two initial experiments are
`shown in Tables I and II.
`
`Genzyme Ex. 1002, pg 48
`
`Genzyme Ex. 1002, pg 48
`
`

`
`9
`TABLE I ,.
`
`4,396,601
`
`EXPERIMENTMB2. KARYOTYPE ANALYSIS ‘OF
`MARROW CELLS o1= IRRADIATED CBA/Ca MICE
`RECEIVING A 1:1 MIXTURE OF CONTROL Ca and
`TRANSFORMED T6T6 MARROW CELLS
`
`5
`
`Karyotype ,
`Duration of Mtx Treatment
`_
`(% T6T6) .
`(days) .
`Recipient‘
`57‘
`,
`0-24
`Primary 1
`59
`0-32
`Primary 2
`79
`32-46
`Secondary 2
`67
`0-39
`Primary 3
`97
`39-53
`Secondary 3a
`93
`39-67
`Secondary 3b
`84
`39-73
`Secondary 3c
`‘Irradiated CBA/Ca recipients of the 1:1 mixture of a Ca transformed with wild
`type DNA and T6T6 cells transformed with 3T6R 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‘-' and bear
`the same identifying number. Karyotype analysis of recipient bone marrow cells
`were perfonned alter the designated interval of methotrexate treatment. Between 50
`and I00 chromosome spreads were analyzed.
`
`10
`
`TABLE II
`EXPERIMENT TV4. KARYOTYPE ANALYSIS OF
`MARROW CELLS OF CBA/Ca MICE RECEIVING A 1:l
`MIXTURE OF CONTROL Ca and TRANSFORMED T6
`MARROW CELLS
`
`Days Without _
`Days with Mtx
`Karyotype
`(% T6)
`Mtx
`Recipient‘
`79
`—
`0-33
`Primary 1
`75
`—
`0-40
`Primary 2
`74
`—
`0-47
`Primary 3
`83
`48-68
`0-47
`Primary 3
`88, 88, 100‘
`-—
`47-61
`Secondary 3
`75
`--
`0-54
`Primary 4
`83
`—’
`54-72
`Secondary 4
`96
`—
`0-65
`Primary 5
`63
`66-113
`0-65
`Primary 5
`‘Irradiated recipients of the 1:] mixture of Ca cells transformed with wild type
`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.
`
`Between roughly days 30 and 40 a clear increase in
`the percentage of bone marrow cells displaying the
`T6T6 markerwas observed in primary recipient ani-
`mals. Marrow from these mice was-injected into irradi-
`ated secondary recipients which were then treated with
`methotrexate. They, too, showed an increased ratio of
`T6T6 to Ca karyotypes, above that seen in the primary
`marrow recipients. Seven such experiments were per-
`formed and this same pattern was seen in five indepen-
`dent experiments involving l9 primary recipient ani-
`mals and 30 secondary recipients. Only two experi-
`ments during this same period failed to show a predomi-
`nance of transformed karyotype.
`When methotrexate treatment of animals receiving
`transformed marrow cells was stopped, the predomi-
`nance of T6T6 karyotypes persisted for at least 3 weeks
`(Primary Recipient 3, Table III) but gradually dimin-
`ished by 8 weeks without treatment (Primary Recipient
`5, Table II).
`
`TABLE III '
`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
`Spleen Colohies
`T6T6
`‘rare
`Ca- Mixed
`(%)
`‘(%)
`(%)
`(%)
`57
`50
`50
`0
`
`Duration of
`Recipient Mtx (days)
`0-24
`Primary 1
`
`20
`
`25
`
`30
`
`35
`
`40
`
`‘
`
`45
`
`50
`
`55
`
`60
`
`65
`
`.
`_« 1
`,
`
`, TABLE III-continued
`KARYOTYPE ANALYSIS OF BONE MARROW AND
`PLURIPOTENT STEM CELLS. FROM CBA/Ca MICE -
`RECEIVING 1:l MIXTURE OF CONTROL Ca AND ‘
`TRANSFORMED T6T6 BONE MARROW CELLS '
`
`Duration of
`Recipient Mtx (days)
`Primary 2
`0-40
`0-47
`Primary 3
`
`Bone Marrow
`Karyotype
`T6T6
`(%)
`75
`74
`
`R
`
`Karyotype of
`Spleen Colonies
`T6T6
`Ca Mixed
`(%)
`(%)
`.
`(%)
`57
`26
`17
`58
`8
`33
`
`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 p.glml for 90 minutes before treatment
`with hypotonic KCL and fixation with acetic acid-
`/ethanol for chromosome spreads.
`_
`In order to analyze whether the predominance. of
`T6T6-marked cells involved pluripotent stem cellsvas
`well as other proliferating marrow cells, marrow was
`taken from selected primary recipient animals and
`5Xl04 cells were injected into irradiated recipient
`CBA/Ca mice in a typical spleen colony-forming
`(CFU-S) assay. (Tell and McCu11och Rad. Res. 142213,
`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 stemcell 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-
`vantage or increased resistance to Mtx and to analyze
`the contribution of endogenous hematopoietic repopu-
`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-
`nance of Ca karyotypes as anticipated from the contri-
`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 TRANSFORME
`T6T6 MARROW CELLS
`Duration of Mtx Treatment
`Karyotype
`
`Recipient‘
`(days)
`(% T6T6)
`Primary 1
`0-33
`31
`Primary 2
`0-40
`24
`Primary 3
`none
`26
`Primary 4
`0-60
`40
`Primary 5
`none
`21
`Primary 6
`0-26
`50
`Secondary 6
`27-48
`28 _
`-Primary 7
`0-53
`56
`Primary 8
`0-56
`57
`Primary 9
`0-42
`40
`Primary 10
`0-56
`24
`
`In primary Ca recipients of equal mixtures of mock
`transformed T6 and Ca cells the percentage of dividing
`marrow cells with the T6 marker varied between 21 and
`
`Genzyme Ex. 1002, pg 49
`
`Genzyme Ex. 1002, pg 49
`
`

`
`4,396,601
`
`12
`
`11
`' 56%. It is presumed that animals with lower percent-
`ages of T6 had restored their hematopoiesis at least in
`part from endogenous Ca cells surviving the irradiation.
`In a final study to demonstrate transformation to drug
`resistance,
`in two independent experiments the usual
`procedure was reversed and Ca cells were transformed
`and T6 cells were used as the controls. After injection
`of a 1:1 mixture of Ca and T6 into irradiated T6 animals
`they were treated with Mtx or left untreated for two
`months.
`
`TABLE V
`EXPERIMENT MB6. KARYOTYPE ANALYSIS OF
`MARROW CELLS CBA/T6T6 MICE RECEIVING A 1:1
`MIXTURE OF CONTROL T6 CELLS AND
`TRANSFORMED Ca MARROW CELLS
`Mtx
`Duration
`Karyotype
`
`Recipien