WO1996039490A1 - Organ function replacement compositions and methods of making and using the same - Google Patents

Abstract

Recombinant cells are disclosed. The cells comprise a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to an activatable regulatory element. The cells additionally comprise a gene that encodes a protein which can be targeted for selective elimination. Methods of treating an individual suspected of suffering from an organ insufficiency condition are disclosed. The methods comprise introducing into such an individual, a plurality of recombinant cells that comprise a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to an activatable regulatory element and that also comprise a gene that encodes a protein which can be targeted for selective elimination. Implantable devices are disclosed. The implantable devices comprise a plurality of recombinant cells that comprise a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to an activatable regulatory element and that also comprise a gene that encodes a protein which can be targeted for selective elimination. Gene therapy compositions and methods are disclosed for preparing and using recombinant cells to deliver therapeutic proteins to individuals.

Description

ORGAN FUNCTION REPLACEMENT COMPOSITIONS AND METHODS OF MAKING AND USING THE SAME

FIELD OF THE INVENTION

The present invention relates to differentiated cells which can be used in transplant procedures for treating individuals suffering from organ dysfunction or failure.

BACKGROUND OF THE INVENTION

Currently, whole organ liver transplantation is the treatment of choice for liver-based metabolic diseases and hepatic failure. Despite growing successes associated with whole organ liver transplantation, surgical risks and complications contribute significantly to patient morbidity and mortality.

In many individuals suffering from liver insufficiency conditions, liver transplantation is not possible either due to the lack of a suitable donor and/or the condition of the patient will not tolerate the trauma of the transplantation procedure. In some individuals suffering from liver insufficiency conditions, a transplantation is not necessary if liver function can be restored or augmented for a period of time sufficient for the individual's dysfunctioning or non- functioning liver to heal, regenerate or otherwise regain function. In such individuals, the introduction and/or use of liver cells can provide or augment liver function. Attempts have been made to develop external artificial livers, referred to as extracorporeal liver assist devices (ELADs) which are connected to the individuals through the circulatory system. An individual's blood flow is diverted to the ELAD which contains cells and tissues within cartridges. The ELAD then functions as the liver.

U.S. Patent Number 4,853,324, which is incorporated herein by reference, describes an organ support system including an ELAD. The ELAD uses hepatocytes that were derived from cultured transformed hepatocytes originally generated by transforming primary hepatocytes with a temperature sensitive SV40 virus and culturing the cells under conditions in which the temperature sensitive virus transforms the cells. After sufficient numbers of proliferating tumor cells are generated, the culture conditions are changed to inactivate the virus and cause the cells to cease having the transformed phenotype and function as normal hepatocytes. The hepatocyte cells are placed in the ELAD cartridge and patient blood flow is directed through the device.

U.S. Patent Number 5,270,192, which is incorporated herein by reference, describes an organ support system including an ELAD that uses primary hepatocytes, particularly non-human hepatocytes. U.S. Patent Number 5,368,555, which is incorporated herein by reference, describes an organ support system including an ELAD that uses hepatoblastoma cells or other tumor cells which are derived from liver cells. Patient blood flow is directed through such devices which function in place of or as an augmentation to the patient's liver. The cells can be cultured and therefore large numbers of cells can be made available. Some transformed liver cells continue to express proteins characteristic of liver cells and these cells may continue to function as liver cells. One shortcoming of such cells is that they may not function as differentiated liver cells despite the continuing expression of a liver cell marker.

Hepatocyte transplantation - that is, liver cell transplantation as opposed to whole organ transplantation - potentially could be used to treat acute liver failure and metabolic diseases not associated with cirrhosis, thus avoiding surgical intervention and its associated risks. Cellular transplants could also be used to palliate patients with chronic liver failure who are awaiting whole organ transplantation.

U.S. Patent Number 4,391,909, which is incorporated herein by reference, describes microencapsulation technology for encapsulating tissue and cells, including hepatocytes, that can then be implanted into the body of individuals. The encapsulated cells can secrete metabolic products into the individuals.

U.S. Patent Number 4,806,355, which is incorporated herein by reference, describes microencapsulation technology for encapsulating tissue and cells that can then be implanted into the body of individuals. The encapsulated cells can secrete metabolic products into the individuals. In particular, implantation of encapsulated islets cells are disclosed.

U.S. Patent Number 4,902,295, which is incorporated herein by reference, describes transplantation of artificial tissue which comprises cells and tissue in a synthetic biocompatible matrix. The artificial tissue is implanted into the body of individuals where it can secrete metabolic product.

U.S. Patent Number 4,942,129 describes dual membrane microencapsulation technology for encapsulating tissue and cells. The encapsulated tissue and cells can then be implanted into the body of individuals and secrete metabolic products. U.S. Patent Number 4,997,443, which is incorporated herein by reference, describes transplantation of artificial tissue which comprises cells and tissue in a synthetic biocompatible matrix. The artificial tissue is implanted into the body of individuals where it can secrete metabolic products.

U.S. Patent Number 5,334,640, which is incorporated herein by reference, describes microencapsulation technology.

U.S. Patent Number 5,314,471, which is incorporated herein by reference, describes tissue implant systems which are membrane assemblies. The implant devices are chambers which are fabricated to contain cells and tissues, which allow for the survival of the cells and which allow for secretion of products.

U.S. Patent Number 5,344,454, which is incorporated herein by reference, describes tissue implant systems which are membrane assemblies. The implant devices are chambers which are fabricated to contain cells and tissues, which allow for the survival of the cells and which allow for secretion of products.

There is a need for an alternative means to provide a patient who is suffering from severe liver insufficiency with the biological capacity equivalent of at least 10-20% of native liver function up to complete restoration of biological capacity. There is a need for an alternative means for treating a patient who is suffering from severe liver insufficiency with effective treatment without exposing the patient to surgical risks and complications that contribute significantly to patient morbidity and mortality. There is a need for an alternative means to provide a patient who is suffering from severe liver insufficiency with effective treatment for acute liver failure and metabolic diseases. There is a need for an alternative to using primary or transformed liver cells to restore or augment liver function in a patient who is suffering from severe liver insufficiency with effective treatment without exposing the patient to surgical risks and complications that contribute significantly to patient morbidity and mortality. There is a need for an alternative to using primary or transformed liver cells to restore or augment liver function in a patient who is suffering from severe liver insufficiency with effective treatment for acute liver failure and metabolic diseases.

Transplantation of other organs generally suffer from similar shortcomings. There is a need for an alternative means to treat a patient who is suffering from severe organ insufficiency to complete restoration of biological capacity. There is a need for an alternative means to treat a patient who is suffering from severe organ insufficiency with effective treatment without exposing the patient to surgical risks and complications that contribute significantly to patient morbidity and mortality. The is a need for an alternative means to provide a patient who is suffering from severe organ insufficiency with effective treatment for acute organ failure and metabolic diseases.

SUMMARY OF THE INVENTION

The present invention relates to recombinant hepatocyte cells. The cells comprise a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to an activatable regulatory element. The cells additionally comprise a gene that encodes a protein which can be targeted for selective elimination. The present invention relates to methods of treating an individual suspected of suffering from a liver insufficiency condition. The methods comprise introducing into such an individual, a plurality of recombinant hepatocyte cells that comprise a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to an activatable regulatory element and that also comprise a gene that encodes a protein which can be targeted for selective elimination.

The present invention relates to implantable devices which comprise a plurality of recombinant hepatocyte cells that comprise a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to an activatable regulatory element and that also comprise a gene that encodes a protein which can be targeted for selective elimination.

The present invention relates to recombinant cells that comprise a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to an activatable regulatory element. The cells additionally comprise a gene that encodes a protein which can be targeted for selective elimination.

The present invention relates to methods of treating an individual suspected of suffering from an organ insufficiency condition. The methods comprise introducing into such an individual, a plurality of recombinant cells of the type from the organ which is dysfunctional. The cells comprise a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to an activatable regulatory element and that also comprise a gene that encodes a protein which can be targeted for selective elimination.

The present invention relates to implantable devices which comprise a plurality of recombinant cells that comprise a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to an activatable regulatory element and that also comprise a gene that encodes a protein which can be targeted for selective elimination.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

As used herein the term, "recombinant expression cassette" refers to an exogenous nucleotide sequence that encode a protein operably linked to regulatory elements necessary for expression of the protein encoded by the nucleotide sequence in a cell.

As used herein the term, "exogenous nucleotide sequence" is meant to refer to a nucleotide sequence which originated from a cell other than the cell that the exogenous nucleotide sequence is in. Exogenous nucleotide sequences may be genomic DNA, cDNA or synthetic DNA that encodes proteins foreign to the individual or that are normally produced by the individual.

As used herein the term, "condition sensitive transforming protein" is meant to refer to a protein which when expressed by a cell and active induces a transformed phenotype. The condition sensitive transforming protein is rendered active or inactive based upon environmental conditions such as temperature, pH, the presence or absence of activator/repressor molecules, media conditions or atmospheric conditions. Condition sensitive transforming proteins are generally rendered active or inactive based upon temperature, the presence or absence of molecules that they form heterodimers with. An example of a condition sensitive transforming protein is a temperature sensitive mutant of SV40 large T antigen. At 33°C, the protein is active and its presence in cells renders them transformed. At 37°C, the protein is inactive and its presence in cells does not otherwise alter the normal untransformed phenotype. Other condition sensitive transforming proteins include, but are not limited to, temperature sensitive mutants of oncogenes, particularly: temperature sensitive mutants of abl , temperature sensitive mutants of avian sarcoma, and temperature sensitive mutants of ras .

As used herein the term, "activatable regulatory element" is meant to refer to promoters which are referred to interchangeably herein as regulatable or inducible promoters. In addition, "activatable regulatory element" is meant to refer to transactivator/repressor elements which control expression of genes operably linked to them. An example of an inducible promoter includes mouse metallotheonein promoter which is an active promoter in the presence of zinc and is inactive in the absence of zinc. Another example of an regulatable promoter includes the promoter which is regulated by tetracycline. An example of a transactivator/repressor element is the TAR region of the human immunodeficiency virus 5' long terminal repeat promoter. The promoter is active in the presence of the HIV protein TAT and is inactive in the absence of TAT. Soluble TAT may be added to culture media to transactivate expression cassettes that include a 5' LTR including TAR region in cells. Another example of a transactivator/repressor element is the REV response element (RRE) of the human immunodeficiency virus which is present on the late messages of HIV env, gag, pol , vif, vpr and vpu . The HIV protein REV, when present, "drags" these messages out of the nucleus and into cytoplasm where the message is translated into protein. In the absence of REV, proteins whose message contains an RRE are not expressed. Proteins whose messages include an RRE are expressed in the presence of the HIV protein REV and not expressed in the absence of REV. Messages which are regulated by RRE further comprise additional coding sequences which are linked to the desired sequence by the RRE. Upon processing by the REV, the desired sequence is liberated from the construct and the mRNA that encodes the sequence is translated. Soluble REV may be added to culture media to transactivate expression cassettes that include RRE on the message. Both RRE and TAR regions are well known and the nucleotide sequences of both transactivation regions are published in the AIDS Los Alamos database. Likewise, the amino acid sequences of HIV REV and HIV TAT are well known and publicly available (See also: NIH AIDS Research and Reference Reagent Program (ARRRP) ) . Another example of a transactivator/repressor element is the lac operon system in the E. coli lactose gene. The promoter is active in the presence of the IPTG and is inactive in the absence of IPTG.

As used herein the term, "protein which can be targeted for selective elimination" is meant to refer to proteins which, when present in cells, can be used to mark or render the susceptible cells to be killed by agents that will not kill cells that lack the protein. An example of a protein which can be targeted for selective elimination is the herpes simplex virus thymidine kinase (HSV tk) gene. The agent gangcyclovir kills cells that have the HSV tk gene.

As used herein, the term "microencapsulation" is meant to refer to the encasement of a cell into a biocompatible covering which enables the cell to live in an individual, secrete products produced by the cell into the individual.

As used herein, the term "implantable device" is meant to refer to a permeable biocompatible container or a biocompatible matrix. An implantable device can be safely implanted into an individual, can hold cells that can survive when implanted into the individual, can allow for nutrients and oxygen to enter the device, and can allow for the products produced by the cells to secrete into the body.

As used herein, the term "liver insufficiency condition" is meant to refer a disease, disorder or injury, including infectious or congenital diseases, characterized by a non-functioning or underfunctioning of the liver. Using various well known tests and observing symptoms, those having ordinary skill in the art such as physicians can readily identify individuals suspected of suffering from liver insufficiency conditions.

As used herein, the term "organ insufficiency condition" is meant to refer to a disease, disorder or injury, including infectious or congenital diseases, characterized by a non-functioning or underfunctioning of a particular organ of the body. Using various well known tests and observing symptoms, those having ordinary skill in the art such as physicians can readily identify individuals suspected of suffering from various organ insufficiency conditions.

The present invention provides the means to grow large amounts of cells that can be safely transplanted into an individual. The present invention provides the means to reversibly transform primary cells in order to generate large numbers of such cells and to revert the phenotypes of such cells to functioning cells of the organ from which they were derived. The cells can be introduced into an individual and maintained in an individual as non-transformed functioning cells. The cells can be engineered to be non-tumorigenic and to have reduced immunogenicity following transplantation in allogeneic recipients. The present invention provides the means to grow large amounts of hepatocytes that can be safely transplanted into an individual. The present invention provides the means to reversibly transform primary hepatocytes in order to generate large numbers of hepatocytes and to revert the phenotypes of the cells to functioning hepatocytes. The hepatocytes can be introduced into an individual and maintained in an individual as non-transformed functioning hepatocytes. The cells can be engineered to be non-tumorigenic and to possibly circumvent rejection following transplantation in allogeneic recipients. In order to stabilize a patient with severe liver insufficiency, the biological capacity of the transplanted hepatocytes would need to be the equivalent of about 10-20% of native liver function (between 100 and 200 gms of cells) for each case. If primary hepatocytes were used to treat each patient, a continuous supply of fresh cells would be needed. Unfortunately, the number of human livers available for hepatocyte isolation is limited by competition for use in whole organ transplantation. Considering the cost of hepatocyte isolation and the need for consistent and functionally uniform cell preparations, it unlikely that human hepatocyte could be isolated on a scale sufficient to treat more than a fraction of the patients who would need them. The use of animal cells would result in additional concerns related to the transmission of infectious agents and immunologic and physiologic incompatibilities between humans and the donor species.

Recombinant hepatocyte cells of the present invention provide the advantage of availability, uniformity and sterility. Further, they can be grown in unlimited quantity and at far less cost compared to isolated primary hepatocytes. The cell line must retain most of the features of a normal hepatocyte, specifically those which contribute to the biological function of the liver.

Since hepatocytes contain numerous metabolic elements, it would be almost impossible to molecularly clone all of these, along with their regulatory elements, into an established cell line. Accordingly, a clonal hepatocyte cell line according to the invention is generated by transducing primary hepatocytes with a transforming gene whose expression or the activity of the expression product is regulatable, thus making the transformation phenotype inducible and repressible. Under induced conditions, the expression of transforming gene and activity of the transforming protein produced thereby results in unlimited growth and loss of differentiated function. Under non-induced conditions, the cells have restricted growth potential and express differentiated hepatocyte characteristics. Thus, the primary hepatocytes are transfected to assume a transformed phenotype under controlled conditions thereby allowing proliferation of the cells. Proliferating cells may then be converted from proliferating cells to differentiated cells by altering the conditions to inhibit the proliferation signal and bring about differentiation into hepatocytic phenotype.

According to the present invention, clonal hepatocytes are used to treat individuals suffering from liver insufficiency. The hepatocytes are derived from primary hepatocyte which have been isolated and into which a recombinant expression cassette is incorporated. The recombinant expression cassette contains an exogenous nucleotide sequence that encodes a condition sensitive transforming protein. The expression of the condition sensitive protein under activating conditions results in the transformation of the cells. In the expression cassette, the expression of the exogenous nucleotide sequence that encodes a condition sensitive transforming protein is inducible or otherwise controllable. Thus, the cells are cultured under conditions which result in expression of the condition sensitive transforming protein and the cells are maintained under conditions in which that protein is active. Thus, the cells assume a transformed phenotype characterized by cell proliferation. The transformed phenotype allows for large numbers of the cells to be grown. When the conditions under which the cells are grown do not induce the expression of the exogenous nucleotide sequence that encodes the condition sensitive transforming protein and the conditions that the cells are maintained in inactivate the condition sensitive transforming protein, the cells revert to untransformed states and differentiate into functioning hepatocytes which may be used in transplantation protocols. Primary hepatocytes may be obtained from donor human livers or resected human liver tissue; hepatocytes may be obtained from animals also. Methods of culturing primary hepatocytes are well known to those having ordinary skill in the art.

The recombinant expression cassette may be used to generate the clonal cell line which can be regulated to induce the cells to proliferate or differentiate requires 1) a coding sequence for a condition sensitive transforming protein under the regulatory control of an inducible promoter or transactivating/repressing regulatory element. Thus the transforming property of the gene can be regulated and the expression of the gene that encodes it can be tightly controlled.

The active form of the condition sensitive transforming protein must be capable of transmuting the phenotype of the cell to a proliferating, transformed cell. Thus, inducing expression of the gene and maintaining the cells under conditions which render the protein active induces transformation of the cell while repressing expression and maintaining the cells under conditions which render the protein inactive brings about the untransformed phenotype characterized by differentiation into an hepatocyte.

According to some embodiments, the recombinant expression cassette comprises a metallotheonein promoter which can be induced to express an operably linked gene by the presence of zinc. In some embodiments, the nucleotide sequence that encodes the condition sensitive transforming protein is under the regulatory control of the E. coli lactose operon. Several elements of the lactose operon have been modified for use in eukaryotic cells to control gene expression. In the E. coli lactose operon, the lac repressor binds to the lac operator, blocking transcription of the lacZ gene. Synthetic inducers such as isopropyl-/3-D-thiogalactoside (IPTG) bind to the lac repressor, causing a conformational change which effectively decreases the affinity of the repressor for the operator. When the repressor is removed from the operator, transcription from the lactose operon can take place. By regulating the expression of a transforming gene/oncogene using the lac operon, transformation would depend on the presence of IPTG to induce the promoter. In some embodiments, the promoter which controls expression of the transforming gene is the HIV 5' LTR promoter which includes the TAR element. Expression of genes under control of this HIV promoter can be controlled by the presence or absence of the HIV protein Tat. The presence of Tat is required for expression. Thus, gene expression can be regulated by the presence or absence of the tat protein. Other inducible promoter systems are well known to those having ordinary skill in the art and can be used to produce expression cassettes useful in the invention.

The recombinant expression cassette comprise a nucleotide sequence that encodes a condition sensitive transforming protein. The condition sensitive transformed protein is active under certain regulatable conditions and inactive in other regulatable conditions. Thus, conditions can be controlled to induce transformation, i.e. cell proliferation or differentiation. According to some embodiments, the gene encodes a temperature sensitive mutant of a transforming protein such that at some temperatures the protein is active while at other temperatures it is not. In some embodiments, the gene encodes a temperature sensitive mutant of SV40 large T antigen in which at temperatures of about 33°C the protein is active and causes transformation while at temperatures of about 37°C it is inactive and thus does not cause transformation. Cells cultured at permissive temperatures and thus having a transformed phenotype can be induced to differentiate by maintaining them at a non-permissive temperature. (Tooze, J. 1981, Molecular Biology of Tumor Viruses, Part II: DNA Tumor Viruses, Cold Sprig Harbor Press, Cold Spring Harbor, N.Y. , which is incorporated herein by reference) .

The nucleotide sequence that encodes the condition sensitive transforming protein is under the control of inducible, transactivating or repressible regulatory elements. By placing the transforming protein under the control of regulatable regulatory elements, the transformation phenotype may be controlled by two mechanisms, thus providing tighter control. In some embodiments, the gene construct used to generate conditionally immortalized primary hepatocytes comprises plasmid or recombinant virus vectors which use multiple regulatory elements to control the expression of the SV40 temperature-sensitive oncogene. In addition, the cells additionally contain an expressible form of a nucleotide sequence that encodes a protein which can be targeted for selective elimination. This allows for the selective and specific targeted elimination of transplanted cells should a cell display a transformed phenotype or there is any other reason or desire to eliminate all transplanted cells.

Recombinant expression cassettes may be introduced into primary hepatocytes by a variety of means such as for example, direct DNA introduction such as DNA transfection, liposome-mediated transfer, DNA transfer using particle bombardment and recombinant viral vector infection. Those having ordinary skill in the art can readily insert DNA constructs into primary cells routinely. In some embodiments, the recombinant expression cassette is a component of a recombinant viral vector. In some embodiments, the recombinant expression cassette is part of a recombinant adenovirus vector. In some embodiments, the recombinant expression cassette is part of a recombinant retrovirus vector. In some embodiments, the recombinant expression cassette is part of a replication- defective retrovirus. In some embodiments, the recombinant expression cassette is a recombinant Moloney murine leukemia virus. In some embodiments, the recombinant expression cassette is delivered to the cells in a transfection protocol using CaP0₄, lipofectin or cytofectin. In some embodiments, the recombinant expression cassette is delivered using asialoglycoprotein receptor (ASGPR) -mediated endocytosis. In some embodiments, the recombinant expression cassette is a retrovirus which is used to infect the primary cells whereby upon infection, the retroviral genome integrates into the chromosomal DNA of the cell. Those having ordinary skill in the art can use one of many well known techniques for introducing recombinant expression cassettes into cells (Sambrook J, Fritsch E, Maniatis T: Molecular Cloning: A Labo¬ ratory Manual. Cold Spring Harbor: Cold Spring Harbor Labo¬ ratory Press; 1989; and Mulligan, R.C. (1993) Science 260:926- 932; which are each incorporated herein by reference) . Upon introducing the recombinant expression cassette into cells, the cells are cultured to induce the transformed phenotype. Depending upon the nature of the recombinant expression cassette, the culturing conditions to induce and maintain the transformed phenotype vary. However, the cell are cultured under such conditions to induce and maintain the transformed phenotype, producing a clonal cell line of proliferating cells.

When cells are to be used in a transplantation protocol, the transformed cells are cultured under conditions which induce differentiation. That is, the conditions are changed to remove the continuing presence of active transforming protein that induces transformation. When the conditions are changed to stop inducing gene expression of the nucleotide sequence that encodes the transforming protein and when the cells are not maintained in conditions in which the transforming protein is active, the cells cease to be transformed and differentiate into hepatocytes which can be used to supplement or replace the liver function in individuals who are deficient. Transplantation protocols include direct injection of cells into the body of an individual. In preferred modes of administration, the cells are injected into the circulatory system, most preferably into the portal or intraportal artery or vein. In some embodiments, the protocols may employ cells grown on polymer scaffolds (See for example Freed, L. E. et al .

(1994) Bio/Technology 12:689-693, which is incorporated herein by reference) contained within permeable containers which allows secreted biological material to be released but which protect the cells from immunological rejection. In some embodiments, the cells are introduced in as microencapsulated cells or within biocompatible matrices or other implantable devises such as for example those described in U.S. Patent Number 4,391,909, U.S. Patent Number 4,806,355, U.S. Patent Number 4,902,295, U.S. Patent Number 4,942,129, U.S. Patent Number 4,997,443, U.S. Patent Number 5,334,640, U.S. Patent Number 5,314,471, and U.S. Patent Number 5,344,454, which are each described above and incorporated herein by reference.

In some embodiments, conditionally immortalized cell lines are genetically altered to reduce their immunogenicity. Cell lines are generated which express no major histocompatibility complex (MHC) class I or class II surface antigens. Hepatocyte lines may also be transfected with an expression plasmid containing the IL-10 gene. MHC-negative cell lines and lines which locally elaborate the immunosuppressive cytokine IL-10 or other immunomodulating cytokines or soluble proteins can be assessed for their ability to circumvent rejection. Immunologically inert hepatocyte cell lines allow transplant recipients to maintain their grafts without the need for chronic immunosuppression to control rejection or with reduced need for chronic immunosuppression to control rejection.

Conditionally immortalized hepatocyte cell line may be produced which allow for high level production of cells that can function as a normal hepatocyte following transplantation. It is contemplated that the characteristics of these hepatocyte cell lines could be enhanced by molecular cloning of genes which encode specific functions that may be deficient in either the cell line or a patient. For example, it is contemplated that molecular cloning could be used to enhance the function of a cell line whose functional capacity to perform metabolic functions was somewhat diminished or it could be used to express an active version of a defective gene such as a blood clotting factor which results in hemophilia. Finally, if long term cultured hepatocyte cell lines could be genetically manipulated to become immunologically inert, long term immunosuppressive medications would be unnecessary to control rejection post-transplantation. According to one embodiment, primary hepatocytes are conditionally immortalize using a virus vector which contains the gene encoding a thermolabile mutant of the SV40 large T antigen {SV40 (ts) } . These cell lines are characterized at the oncogene permissive (33°C) and non-permissive temperature (37°C) to determine their growth rate, oncologic potential, level of differentiated function and morphology. These experiments establish the degree to which conditionally immortalized hepatocytes function in vi tro as normal hepatocytes. The conditionally immortalized cell lines are grown up in large volumes as transformed cell and then maintained in conditions which repress transformation and result in differentiation. The cells are then used in transplantation protocols for the treatment of liver-based metabolic deficiencies and liver failure.

While the present invention relates primarily to the conditional immortalization of hepatocytes and the use of such cell in transplantation protocols to treat individuals suffering from liver deficiencies, it is contemplated that the methods of the present invention may be applied in the treatment of diseases, conditions and injuries resulting in the absence or reduction in function of other secretory organs. Examples of such organs include the liver, pancreas and organs of the endocrine system, such as the parathyroid, which produce and secrete hormones and growth factors. There are many examples of diseases, disorders or conditions which result in a secretory organ ceasing to function or functioning at a level lower than necessary. For example, diabetes results in the dysfunction of cells of the pancreas which produce insulin. The lack of insulin results in an inability for the body to regulate levels of blood sugar and can result in death. According to the present invention, primary islet cells can be isolated, transfected with an expression cassette of the invention, induced to a transformed phenotype, cultured to produce large numbers of cells which can then be induced to re- differentiate and be transplanted as functional islets. Neurons and glial cells may be transformed according to the invention to generate large numbers of differentiated cells for transplantation.

According to the present invention, primary cells of the type for which an individual is experiencing a reduction or absence in function are transfected with constructs comprising an regulatable promoter linked to a gene which induces transformation. The cells are isolated as primary cells and transfected with a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to an activatable regulatory element. The cells additionally comprise a gene that encodes a protein which can be targeted for selective elimination. In some preferred embodiments, the expression cassette includes both an inducible promoter and a transactivatable/repressible regulatory elements which regulate expression of the a condition sensitive transforming protein. The expression cassette further includes a gene which allows for the selective elimination of such cells.

According to some embodiments of the present invention, primary cells are transfected with constructs comprising inducible promoter linked to a gene which induces transformation. The cells are isolated as primary cells and transfected with a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to an activatable regulatory element. The cells additionally comprise a gene that encodes a protein which can be targeted for selective elimination. In addition, proteins such as insulin, other growth factors, and other cytokines may be produced in such vectors. In some preferred embodiments, the expression cassette includes both an inducible promoter and a transactivatable/repressible regulatory elements which regulate expression of the a condition sensitive transforming protein. The expression cassette further includes a gene which allows for the selective elimination of such cells. The cells further comprise a therapeutic gene operably linked to regulatory elements that are functional in the cells into which the gene is transfected.

According to preferred embodiments of the invention, an expression cassette under the regulatory control of the lac operon include an HIV 5'LTR with a TAR sequence operably linked to a coding sequence that includes an SV40 temperature sensitive T antigen linked to an RRE linked to an HIV polymerase gene and a polyadenylation signal. In addition, an H S V t k g e n e i s p r o v i d e d . The cells are transplanted into individuals for whom the therapeutic protein is desirable for treatment of a diseases or disorder. The protein is expressed in therapeutically effective amounts.

EXAMPLES EXAMPLE 1

We have developed procedures that permit isolation of conditionally immortalized cells of several origins, including hepatocytes. The method uses a variety of virus and plasmid constructs which express temperature-sensitive versions of the SV40 T antigen {SV40(ts)}; this allows for the reversion of the tumorigenic phenotype in vi tro by a temperature shift. When cultured at 37-39°C, SV40(ts) transformed hepatocytes cease to proliferate, have markedly reduced DNA synthesis, and develop morphologic characteristics of differentiated hepatocytes.

Generation and characterization of SV40 (ts) transformed hepatocyte cell lines .

We immortalized primary Gunn rat hepatocytes by transduction with a recombinant Moloney murine leukemia virus expressing the thermolabile mutant SV40 (tsA58) . After culturing at the permissive temperature (33°C) , colonies emerged in 3 weeks. Individual colonies were then picked and subcloned. By light microscopy, immortalized hepatocyte clones exhibited a transformed phenotype at 33°C, but developed morphologic characteristics of differentiated hepatocytes at 39°C. In addition, at 39°C, transmission electron microscopy demonstrated peroxisomes and intercellular junctions with microvilli characteristic of bile canaliculi.

The incorporation of ³H-thymidine by the immortalized cells cultured for 24 hrs or 48 hrs at 33°C was 25 to 50 fold greater than that by primary hepatocytes cultured at 37°C. When cultured at the non-permissive temperature (39°C) , DNA ³H- thymidine incorporation was reduced to 2 to 2.5 fold that of primary hepatocytes. At 33°C, the immortalized cells doubled in number in approximately 72 hrs, however, there was no significant increase in cell number when the immortalized cells were cultured at 39°C or when primary hepatocytes (plated 20 hrs earlier) were cultured at 33°C or 39°C.

In order to measure protein expression or cellular function, cells were cultured at 33°C or 37°C after initial incubation for 4 hrs at 39°C to degrade the SV40 (ts) . Immunotransblot studies showed that immortalized cells, cultured at 33°C, contained albumin, asialoglycoprotein receptors (ASGPR) and androsterone-UGT at approximately 5-10% of the levels found in primary hepatocytes cultured for 24 hrs under the same conditions. In contrast, glutathione-S- transferase Y_p (GSTY_p) , an oncofetal protein, was expressed at a 10-times higher level. When cultured at 37°C, cellular concentrations of albumin, ASGPR and androsterone-UGT increased to 25-40% of the level in primary hepatocytes while GST-Y_p concentration decreased markedly. Northern blot analysis with probes specific for albumin and androsterone-UGT showed that the cellular concentration of these mRNAs paralleled the changes in the concentration of the corresponding proteins.

The function of asialoglycoprotein receptors (ASGPR) in conditionally immortalized hepatocytes was also evaluated. Internalization of texas-red-labeled asialoorosomucoid (ASOR) was demonstrated by the finding of fluorescence-labeled ASOR within the cultured cells. Cells cultured at 37°C showed internalization of a large number of fluorescent vesicles, which was mostly abolished when uptake was performed in the presence of excess unlabeled ASOR. Cells cultured at 33°C demonstrate a greatly reduced number of fluorescent vesicles compared to those cultured at 37°C. Binding of ¹²⁵I-labeled ASOR by conditionally immortalized hepatocytes cultured at 33°C was approximately 30% of that by cultured primary hepatocytes. At 37°C, the binding increased to approximately 55% of the level in primary hepatocytes. Internalization and degradation of ASOR was determined by the appearance of acid soluble breakdown products of the bound ASOR after 2 hrs of incubation. Cells cultured at 33°C degraded ASOR at approximately 23% the rate observed in primary hepatocytes. In cells cultured at 37°C, the rate of ASOR degradation was approximately 54% that in primary hepatocytes.

MHC gene expression was also studied by flow cytometry using monoclonal antibodies (MoAb) to MHC class I and class II surface antigens. Conditionally immortalized hepatocyte clones, grown at 33°C, did not express MHC class II antigens but expressed MHC class I antigens at the same level as primary rat hepatocytes.

Introduction of new genes into SV40 (ts) transformed hepatocyte clones . Recombinant retrovirus vectors and plasmids capable of expressing the E. coli _/3-galactosidase gene (lacZ) in eukaryotic cells were used to transduce conditionally immortalized hepatocytes. Transduction efficiency was assessed using 5-bromo-4-chloro-3-indolyl _/S-D-galactoside (X-gal) cytochemical staining. Both retrovirus infection (virus titer 5 x 104 cfu/ml) and plasmid transfection transduced approximately 10% of the cultured cells.

In order to increase the number of cells which expressed the introduced new gene, cells were stained with fluorescein di-S-D-glactopyranoside (FDG) . Because FDG is cleaved by _/S-galactosidase in lacZ⁺ cells to yield fluorescein, cells which express _/S-galactosidase can be detected by fluorescence-activated cell sorting (FACS) without fixation. This technology for _/S-galactosidase detection is more sensitive than any other available cytochemical or biochemical method. Cells were analyzed with a FACStar Plus cell sorter, and selected based on a predetermined fluorescence intensity threshold. Greater than ninety-five percent of selected cells expressed _/S-galactosidase as assessed by X-gal cytochemical staining and were used in preliminary transplant experiments. Because of cellular variation in _/β-galactosidase expression, individual cells have been cloned into 96-well plates and clones are being individually screened for expression by flow cytometry and X-gal cytochemical staining.

Transplantation of SV40 (ts) transformed hepatocytes in rodents . Conditionally immortalized hepatocyte clones which express the SV40 nuclear T antigen at 33°C, but not at 37-39°C, when assessed by fluorescence staining, have been used in transplantation experiments. When 10 - 50 x IO⁶ cells were transplanted into the spleens of syngeneic rats, clusters of hepatocytes were easily identified in the white pulp of the spleen and appear morphologically to have a normal cytoplasm to nucleus ratio. Abnormal hepatocytes could not be identified in the liver of these animals and no tumors have been found in transplanted syngeneic rats or transplanted mice with severe combined immunodeficiency syndrome (SCID) at one and 3 months post-transplantation.

LacZ transduced, conditionally immortalized hepatocytes were then transplanted into syngeneic rat spleens in order to determine their trafficking pattern. The vast majority of intrasplenically transplanted hepatocytes were found in the liver when tissues were histologically stained with X-gal and counterstained with hematoxylin and eosin. Both intrasplenically and intraportally transplanted cells assimilated into hepatic cords.

Most experiments were done using cultured cells which were placed at 39°C for 4-6 hrs prior to transplantation. However, when cultured hepatocytes were transplanted directly from 33°C, the number of cells which could be identified on liver and spleen sectioning was estimated to have increased two- to three-fold. Thus, the transplanted hepatocyte mass significantly increased in vivo as a result of the delay in oncogene down-regulation. This characteristic may enhance our ability to provide adequate hepatic function, and correct deficiencies using transplanted hepatocytes.

Use of recombinant vectors containing conditional promoters to regulate expression of the SV40 (ts) oncogene . The human immunodeficiency virus (HIV) encodes several regulatory proteins that are not found in other retroviruses. The tat protein, which is one of these regulatory proteins, is essential for efficient expression from the HIV long terminal repeat (LTR) (Cullen, B.R. 1991 FASEB 5:2361-2368, which is incorporated herein by reference) . Tat mediates this effect by interacting with a segment of the R region in the 5' LTR, termed the TAR element (for trans-activating response) . Experimentally, it has been shown that tat protein can be taken up by cells and trans-activate the HIV-1 promoter (Frankel and Pao, 1988 Cell 55:1189-1193, which is incorporated herein by reference) . Because expression is dependent on tat, the HIV-1 promoter provides a way to introduce a foreign gene into cells where is expressed only when induced. This strategy forms the basis for constructing a vector capable of regulating the expression of the temperature-sensitive SV40 oncogene.

We constructed a plasmid containing the HIV-1 promoter, the SV40(ts), and a poly A region on a bluescript plasmid backbone. This plasmid was then transfected into primary rat hepatocytes. In preliminary studies, we have found that, when cultured at 33°C in the presence of HIV tat protein, at a concentration at 10 μg/ml, primary rat hepatocyte were capable of generating immortalized cell clones which stopped growing within 5 days of removing tat protein from the culture media. Example 2

Hepatocytes are isolated by in si tu collagenase perfusion of livers from Lewis rats. Cells are then suspended in DME (Gibco) containing 4% fetal calf serum, 0.2μM Dexamethasone (Sigma) , penicillin and streptomycin, and plated on Primaria tissue culture flasks (T25) at 4 x IO⁶ cells per flask. Cells are then incubated at 37°C in a 95% air/5%C0₂ atmosphere for 24 hrs. Culture media is then replaced with viral supernatants from Ψ 2-pZipSVts58A cells, which produce an ecotropic, replication-defective retrovirus containing a temperature- sensitive mutant of SV40 T antigen and a gene encoding neomycin resistance. Supernatant containing the recombinant retrovirus is harvested from confluent plates of producer cells 18 hrs after the addition of fresh media and filtered through 0.45 μM filters. Hepatocyte cultures are infected with 3 ml of viral stock per flask in the presence of 12 μg of polybrene (Aldrich Chemical Co.) at 37°C for 4 hrs. The virus-containing media is then aspirated and cultures are maintained in DME containing 4% fetal calf serum, 0.2μM Dexamethasone, penicillin and streptomycin at 33°C. Seven days after retroviral infection, neomycin resistant hepatocytes are selected by adding the neomycin analogue G418 (Gibco) to the culture medium at a final concentration of 400 μm/ml. Based on preliminary data, G418- resistant colonies emerge in 3 weeks; individual colonies are isolated using cloning rings, released by trypsinization, and expanded by culturing at 33°C. Immortalized cell lines are screened for proteins that are preferentially expressed by differentiated hepatocytes using immunotransblot studies. Immunotransblot studies are performed using specific antibodies against three hepatocyte- specific proteins: rat serum albumin, rat ASGPR, and rat androsterone-UGT. To confirm these data, total cellular RNA is extracted, resolved by electrophoresis on 1% agarose gels and blotted on nitrocellulose membranes (Schleicher and Schuell),. Blots are hybridized with cDNA probes for rat serum albumin and rat androsterone-UGT. Clones that contain the highest level of these proteins are chosen for further characterization. In addition to the hepatocyte-specific markers, colonies are analyzed for rat glutathione S- transferase isoform Y_p (GST-Y_p) .

Hepatocytes are also analyzed for asailoglycoprotein receptor expression and function. MHC class I and class II expression and the ability to induce expression with _y- interferon (IFN-_γ, Amgen Biologicals, Thousand Oaks, CA) are determined by flow cytometry. Cell growth is examined by cell counting and DNA turnover is assessed by ³H-thymidine incorporation. METHODS: Animals : Inbred male Lewis rats (150-250 g) are obtained from a Harlan Sprague-Dawley (Indianapolis, IN) . Rats are maintained on standard laboratory rat chow on a 12 hr light/dark cycle.

Recombinant retrovirus and producer cell line: A Ψ -2 cell line that produces a recombinant retrovirus containing the genes encoding a temperature-sensitive SV40 large T antigen (tsA58) and neomycin phosphotransferase (Neo R) was provided by P.S. Jat (Ludwig Institute of Cancer Research, London, UK) . This producer line provides a viral titer of 5 x IO⁴ neomycin resistant CFU per ml when assayed on NIH 3T3 cells (Jat et al , SUPRA) .

Liver cell isolation : Liver perfusion is carried out with Hank's Balanced Salt Solution (HBSS) supplemented with 25mM NaHC0₃ and 2mM EDTA in si tu under light anaesthesia. The portal vein is cannulated and perfused using collagenase type IV (Sigma, 60 mg/150 ml buffer solution) at 20-25 ml/min with a peristaltic pump. After 20 mins HBSS supplemented with 25mM NaHC0₃ is used to wash the system for 3-5 mins. The liver is excised, cut in small pieces and filtered through 4 layers of gauze. The remaining liver fragments are incubated in 10 ml

EDTA containing HBSS at 37°C for 5 minutes and then washed three times with EDTA-free HBSS. After centrifugation through

Percoll 400, the isolated hepatocytes are counted and cultured

(Berry and Friend, 1969 J. Cell Biol . 43:506-530, which is incorporated herein by reference) .

Jmrπunotrans-lot studies: Immortalized hepatocytes are grown to 75% confluence at 33°C. One group is maintained at 33°C for another 20 hrs while a second group is transferred to 39°C for 4 hrs to degrade the intracellular SV40 (ts) , and then cultured at 37°C for 16 hrs. Cells are released using the non-enzymatic cell dissociation solution, washed with 0.25 M sucrose in 20 mM Tris-HCl, pH 7.4, containing 1 mM EDTA, and collected after centrifugation. Cells are then resuspended in the wash buffer at approximately 10 mg protein/ml and homogenized in a glass/teflon homogenizer. Protein concentration is determined in each aliquot and homogenates, containing 100 μg protein, are subjected to sodium dodecylsulfate (SDS)/10% polyacrylamide gel electrophoresis and electroblotted to polyvinylidene diflouride membranes (Millipore Corp., Bedford, MA) . Immunotransblot studies are performed using antibodies against rat serum albumin, rat ASGPR, rat androsterone-UGT and rat glutathione S-transferase isoform Y_p (GST-Y_p) .

Evaluation of ASGPR function : ASGPR-directed internalization of Texas red-labeled asialoglycoprotein is performed as follows: Asialoorosomucoid (ASO) (Sigma, St. Louis, MO) is conjugated with Texas red. Hepatocytes are plated and grown on glass cover slips at 33°C until approximately 60% of the cover slip surface is covered. Cover slips are then divided into two sets, A and B, and each set is divided into four groups (group 1 through group 4) . Set A is kept at 33°C, while Set B is cultured at 39°C for 2 hrs and then at 37°C for 16 hrs. After this, both are transferred to 4°C. For groups 1 and 3, Texas red-labeled ASO is added to the culture medium at 1 μg/ml. For groups 2 and 4, Texas red-labeled ASO and unlabeled ASO is added to the culture medium at 1 μg/ml and 10 μg/ml, respectively. ASO is allowed to attach to receptors by incubation for two hrs at 4°C, after which the unbound ASO is removed by extensive washing. In order to analyze internalization of the receptor-ligand complex, groups 3 and 4 are transferred to 37°C for 10 min. All samples are then washed with PBS and fixed in 3.5% paraformaldehyde in PBS at 4°C overnight. The fixed cells are then washed with 20mM Tris- HCl, pH 7.4 containing 150mM NaCI and 2mM calcium chloride, and mounted on glass slides with fluorescence mounting medium.

To further assess functional endocytosis via the ASGPR in immortalized hepatocytes, degradation of ASO following receptor-mediated endocytosis is evaluated. In brief, cells are grown at 33°C to an approximate density of 2 x IO⁶ per 65 mm diameter plate. One group is incubated at the permissive temperature (33°C) for 20 hrs while a second group is incubated at 39°C for 2 hrs before culturing at 37°C for 16 hrs. Duplicate plates are then incubated with ¹²⁵I-asialoorosomucoid for 30 min at 4°C for surface binding without internalization. Unbound asialoorosomucoid is removed by washing in ice-cold PBS containing 2mM CaCl₂. From one set of plates, cells are released by scraping with a rubber policeman, and bound radioactivity is determined in a gamma counter. To the other set of plates, 1 ml of culture medium (DME with 4% fetal calf serum) is added and the cells are incubated at 37°C for 2 hrs. After this incubation, cells are precipitated in 10% ice-cold tricholoacetic acid and assessed for radioactivity. Cell morphology: Selected hepatocyte clones, cultured at 33°C or 39°C, are examined by light microscopy and transmission electron microscopy. Cytochemical staining for catalase is performed in order to visualize peroxisomes.

³H-Thymidine uptake : Immortalized hepatocyte clones are cultured for 24 hrs or 48 hrs on Primaria (Becton Dickinson Labware, Lincoln Park, New Jersey) plastic tissue culture plates at 33°C or 39°C at densities of 3 x IO⁶ cells per plate (100 mm diameter) . Cells are incubated with ³H-thymidine (4 μmol/ml containing 3μCi per 35 mm plate) at 37°C for 60 min. ³H-thymidine incorporation by primary hepatocytes is studied under the same conditions for comparison. The labeling medium is removed, the cells are washed with PBS and released using a non-enzymatic cell dissociation solution (Sigma, St. Louis, MO) . Cells are then counted and trichloroacetic acid precipitable radioactivity is determined by scintillation counting using Hydrofluor (National Diagnostics, Manville, N.J.) .

Determination of cell number: Immortalized hepatocyte clones are plated at a density of 2 x IO⁶ cells per plate (100 mm diameter) and cultured at 33°C or 39°C. At 24 hr intervals, cells are released by treatment with the cell dissociation solution described above and cell counts are determined using a hemocytometer. Flow cytometry: The following monoclonal antibodies (provided by Dr. James Markmann, Dept. of Surgery, U. of Pennsylvania) are used: 0X6 and 0X17, which identify framework epitopes of the rat MHC class II Rt. l.B and Rt I.D molecules, and 0X18 and 1-169, which are specific for non-polymorphic and polymorphic determinants of Rat MHC class I molecules, respectively. FITC- conjugated goat F(ab')₂ anti-mouse IgG (Cappell Labs, Cochranville, PA) is used as a secondary antibody. Example 3 Conditionally immortalized hepatocyte clones which exhibit highly differentiated function in vi tro are transduced to express _/S-galactosidase by lipofection using a lacZ containing plasmid construct. Transfected cells are subcloned by fluorescence staining with FDG and sorted into 96- well plates. Individual clones are assessed for β- galactosidase expression by X-gal cytochemical staining. Immunotransblot and ASGPR studies are repeated and clones which continue to demonstrate differentiated hepatocyte characteristics are used for transplantation studies. Ten to 50 million conditionally immortalized hepatocytes are transplanted into the spleens of allogeneic Nagase (NAR) analbuminemic rat recipients. Following transplantation, survival and function of transplanted hepatocytes are monitored by serial immunoassay of serum albumin in the recipient animals. At various time points following transplantation recipient rats are sacrificed to histologically analyze the presence and function of transplanted cells. Liver and spleen tissue are examined for the presence of transplanted hepatocytes by staining with X-gal (which stain donor hepatocytes blue) followed by counterstaining with hematoxylin and eosin. Because β- galactosidase can be stained in living cells with the fluorescent dye FDG, individual hepatocytes are recovered from the livers and spleens of transplanted recipient animals, prepared as a single cell suspension, and sorted to separate host and donor hepatocytes. Donor and host hepatocytes are assayed, as previously outlined, to determine whether donor, conditionally immortalized hepatocytes, function differently than primary rat hepatocytes, when both are recovered directly from recipient rats in the same way. Immunocytochemical examination of liver and spleen tissue is also performed to visualize albumin-containing hepatocytes, while spleen tissue is examined immunohistochemically for hepatocytes which express transferrin, glutamine synthetase and pyruvate kinase.

Acute liver failure is induced in syngeneic Lewis rats by 90% hepatectomy. Following the hepatectomy, between 10 and 50 x 10^s immortalized hepatocytes, cultured at 33°C only or transferred to 39°C for 4 hrs, are transplanted into the spleen. Animals are assessed for survival following this procedure, and survivors are studied long term, as outlined above for hepatocyte persistence and function. Transplantation experiments in syngeneic recipients provide information regarding the growth characteristics, potential for tumor formation, and trafficking patterns of immortalized hepatocyte clones in recipients who are not capable of rejection these grafts. Since tissue culture of primary hepatocytes just prior to transplantation has been shown to delay graft rejection for up to six weeks, allograft rejection is unlikely to limit the ability to assess the function of conditionally immortalized hepatocyte lines in vivo. However, if graft function deteriorates before six weeks, or if hepatocyte grafts do not function, recipient rats are immunosuppressed with Cyclosporine in the form of powdered cyclosporin, dissolved in olive oil

(lOmg/ml) and injected at 30 mg/kg subcutaneously for 7 days.

As an alternative, donor hepatocytes may be treated with Ultraviolet-B (UV-B) irradiation to control graft rejection. UV-B irradiation and short term culture has been shown to modify the immunogenicity of primary islet and hepatocyte transplants. UV-B irradiation (600J/m²) of cultured rat hepatocytes, prior to intraportal infusion has been shown to prolong allograft function in histoincompatible metabolically deficient rats almost indefinitely. METHODS: Animals : Genetically analbuminemic rats (Nagase, NAR) may be obtained from the Special Animal Core of the Liver Research Center of the Albert Einstein College of Medicine. These animals are homozygous for a recessive mutation of the structural rat albumin gene and are characterized by extraordinarily low albumin levels (0.025 to 0.05 mg/ml) and hyperlipidemia. Total serum protein levels are normal because plasma proteins such as transferrin and fibrinogen increase compensatorily to maintain a colloid osmotic pressure gradient which is almost normal. β-galactosidase containing vectors : The BAG plasmid (Price et al . 1987 Proc. Natl . Acad. Sci . USA 84:156-160 which is incorporated herein by reference) , which contains the β- galactosidase gene driven by a MoMLV 5' LTR and a neomycin resistance gene driven by the SV40 early region promoter was provided by Dr. Stephen Goff (Dept. of Biochemistry, Columbia U) . The _/S-galactosidase containing amphotropic retrovirus producer line, 7525 (Sanes, et al . 1986 EMBO J. 5:3133-3142, which is incorporated herein by reference) , which also contains a gene encoding neomycin resistance was provided by Dr. Gary Nabel (Dept. of Medicine, U. of Michigan) .

Induction of Liver Failure: Fulminant liver failure is induced surgically using 90% hepatectomy. This is accomplished as follows: The median and left lateral liver lobes are removed by ligation. The inferior portion of the right lobe is drawn anteriorly and to the left to free its posterior attachment to the diaphragm. The lobe is then removed with two ligature to clear the parenchyma posterior to the vena cava without causing caval compression, leaving only the caudate lobe. Body temperature is maintained in an incubator which provides an environmental temperature greater than 31°C and animals are supported with 20% glucose drinking water. In our laboratory, this procedure results in a 50% mortality in 48 hrs and a 70% mortality in 4 days. Acute liver failure may be induced with D- galactosamine hydrochloride (Sigma, St. Louis, MO), injected intraperitoneally at 2.5-3.5 g/kg. The galactosamine is dissolved in sterile water and adjusted to pH 7.3 with 5 M sodium hydroxide immediately before injection. Animals are supported post-injection as outlined for 90% hepatectomy. This procedure leads to a 75% mortality within 72 hrs. Hepatocyte transplantation : Animals are anesthetized using intraperitoneal injections of pentobarbital. A small surgical incision is made in the animal's flank and the spleen is exposed. Hepatocytes are injected into the inferior pole of the spleen using a 0.14mm OD needle connected to a TB syringe. The blood flow in the splenic artery and vein are temporary occluded to avoid passage of cells into the vena cava during transplantation. Hepatocytes are transplanted directly from 33°C or transferred to a 39°C incubator for 4-6 hrs to down- regulate the SV40(ts) . Serum albumin determination : Serum proteins are resolved by SDS/polyacrylamide gel electrophoresis and electroblotted. A monospecific polyclonal anti-rat albumin (rabbit) anti-serum, with a titer of 1:5000 for immunotransblot experiments was developed for immunoassay of serum albumin (provided by Dr. J. Roy Chowdhury, Liver Research Center, Albert Einstein College of Medicine, Bronx) and is used with ¹²⁵I-staphylococcal protein A to visualize albumin bands. Albumin is then quantified by densitometry of auto radiographed bands. Routine measurement of serum albumin by conventional techniques results in falsely elevated albumin values.

Immunohistochemistry: Immunocytochemical examination of the liver and spleen is performed on eight-micron thick sections. Primary antibodies include the above cited anti-rat serum album (rabbit) antiserum and others provided by Dr. Dahn Clemens (U. of Nebraska, Liver Study Unit, VA Medical Center, Omaha, NE) . The avidin-biotin-peroxidase system is used to localize the attachment of these antibodies.

Northern blot analysis : Total RNA is isolated by homogenation in 4M Guanidium thiocyanate, followed by ultracentrifugation through a 5.7 M CsCl cushion. RNA is then electrophoresed in a 1% agarose-formaldehyde gel, transferred to nitrocellulose and hybridized with the appropriate ³²P-random primer labeled probes.

Identification of β-galactosidase containing hepatocytes : Cells transduced to express lacZ are stained with FDG in 0.1 ml of staining medium {phosphate-buffered saline (PBS)/l0mM Hepes, pH 7.3/4% fetal bovine serum} mixed with an equal volume of prewarmed (37°C) 2mM FDG in water for one minute. FDG loading is stopped by returning the cells to isotonic conditions by adding 0.5 ml of ice-cold staining medium with one μg of propidium iodide per ml. Cells are then analyzed and sorted on a FACStar Plus cell sorter based on a predetermined fluorescence intensity threshold.

Eight-micron thick frozen sections of liver and spleen are fixed in 1.25% glutaraldehyde and stained overnight in X- gal (Stratagene) and counterstained with hematoxylin and eosin as a qualitative confirmation of FACS data.

Blood Testing: Blood glucose and liver tests are monitored approximately 1-2 times per week. This is accomplished through tail vein bleeding. EXAMPLE 4

Survival of hepatocytes transplanted into histoincompatible recipients is limited by allograft rejection. Hepatocytes that are not immunologically activated primarily express MHC class I antigens. Antigen-presenting cells (APC) express MHC class II antigens, are carried in transplanted cells and organs, and present MHC class I antigens in an MHC- restricted fashion to host lymphocytes. Presentation of MHC class I antigens by donor APC results in a cascade of cell- mediated immune interactions that leads to allograft rejection. Modulation or elimination of donor APC or dendritic cells has successfully been used to prolong allograft survival. In addition, using transgenic mice as the source of donor tissue, pancreatic islet cells deficient in the expression of MHC class II antigens have a modest improvement in survival compared to controls, while islet cells deficient in MHC class I expression survive indefinitely, when transplanted into allogeneic recipients. Based on these data, down-regulation of MHC gene expression in conditionally immortalized hepatocytes should affect their susceptibility to rejection when transplanted into allogeneic recipients.

Since the conditionally immortalized hepatocytes are derived from a single cell, it is unlikely that they are able to function as APC, especially since, based on preliminary studies, they do not express MHC class II antigens. They do however express MHC class I antigens on their cell surface. Therefore, conditionally immortalized hepatocyte clones may be rendered MHC cell surface antigen-negative using ethyl- methanesulfonate (EMS) . Hepatocytes are grown to a density of 2 x IO⁵ cells/ml in 25 ml of a 1:5000 dilution of EMS in RPMI containing 10% fetal calf serum (FCS) for 18 hrs. After washing three times in DMEM/2%FCS, cells are allowed to recover in RPMI/10%FCS for 48 hrs. Hepatocytes expressing MHC class I antigens on their cell surface are removed by binding to M450-magnetic beads (Dynal) coated with the anti-rat MHC class I antibody 0X18. MHC negative cells are cloned by limiting dilution and subclones which are consistently negative for MHC class I by flow cytometry are used for transplant experiments.

Conditionally immortalized hepatocyte clones are also transduced to express the immunosuppressive cytokine IL-10.

IL-10 is a product of T_H2 cells (the CD4+ helper T cells which appear to augment antibody production) which inhibits the cytokines produced by simulated T_H1 cells (the CD4+ helper T cells which appear to be responsible for allograft rejection and delayed type hypersensitivity) . It is a potent inhibitor of monocyte-macrophage activation, and inhibits production of tumor necrosis factor alpha (TNF-α;) , IL-1 and also IFN__γ. IL- 10 inhibition of IFN._γ production is primarily due to its ability to block production of the IFN._γ_inducer, IL-12, from accessory cells. IL-10 has been shown to inhibit the ability of listeria-infected macrophages to drive T_H1 cell differentiation, most likely through its effects on IL-12 production. In addition, local expression of either the TGF-_/S or IL-10, following the direct injection of these genes, has recently been reported to prolong the survival of non- vascularized cardiac allografts by more than two-fold in mice. Conditionally immortalized hepatocyte cell clones are transduced by transfection with a plasmid containing the IL-10 gene driven by the SV40 early region promoter and, for selection purposes, a second expression plasmid containing the dihydrofolate reductase gene. Cells are grown in 0.25μM methotrexate (Sigma) and subcloned by limiting dilution. Methotrexate resistant colonies are then assayed for IL-10 production.

Transduction with IL-10 may affect some of the important differentiated functions of hepatocyte cell lines. Therefore, following transfection and screening, clones are analyzed for differentiated function prior to their use in transplantation experiments. Cells which continue to demonstrate differentiated hepatocyte function and express IL- 10 in vi tro are used in transplant experiments as outlined. Post transplantation, spleen sections are analyzed for IL-10 production by transplanted hepatocyte using immunohistochemistry.

If the protocol for altering MHC class I antigen expression in hepatocyte cell lines is not successful, the expression of these genes may be altered by genetically disrupting the expression of the _/S2-τnicroglobulin gene. MHC class I heavy chains are noncovalently associated with the β2 - microglobulin light chain on cell surfaces. Disruption of the /82-microglobulin gene causes class I heavy chains to accumulate in the endoplasmic reticulum with resultant loss of MHC class I cell surface expression. A recombinant plasmid, which contains an antibiotic resistance gene inserted into the coding region of the _/S2-microglobulin gene (creating a mutation) , flanked by two long regions of complete homoiogy, and the herpes simplex thymidine kinase (HSVtk) gene placed at the end

(outside the region of homoiogy) was constructed specifically for this purpose. This plasmid was transfected into ES cells to create an MHC class I "knockout" mouse. Upon transfection, the recombinant plasmid DNA undergoes homologous recombination in the two flanking regions so that the interior portion of the _/82-microglobulin gene is replaced by the mutated part of the gene. Positive/negative selection is used to isolate cells which have successfully undergone recombination. The antibiotic resistance gene is used to positively select clones containing the mutated _/82-microglobulin gene and cells are grown in gancyclovir to select against cells carrying the HSVtk gene, which should not be integrated. ES cells are used to create heterozygous mice which are later mated to produce homozygous offspring. In order to create MHC mice class I "knockout" clonal cell lines, two cycles of homologous recombination are required in order to mutate both β2 - microglobulin alleles. This requires the use of two plasmids, each containing a different antibiotic resistance marker. METHODS:

Plasmids : Plasmids containing human and mouse cDNAs for IL-10 and IL-4 were generously provided by Dr. Tony Troutt

(Queensland Institute of Medical Research, Brisbane,

Australia) . These cDNAs were cloned into pCD-based expression vectors at DNAX Research Institute, Palo Alto, CA. The TGF-_/8 expression vector was provided by Dr. Jonathan Bromberg (Dept. of Surgery, Medical U. of South Carolina) .

Transfection : Cells are transfected using either lipofection (Lipofectin Reagent, BRL Gibco, Gaithersburg, MD) or the CaP0₄ (Stratagene, La Jolla, CA) method. Selection for cells which have been stably transfected is accomplished by co- transfection, at a ratio of 5-10:1, with a second plasmid expressing a drug resistance gene (hygromycin, methotrexate, neomycin, etc.) . Measurement of IL-10 : IL-10 is assayed using a two-antibody capture ELISA. Ninety-six well flat-bottomed plates are coated with 50 μl of antimouse IL-10 (Pharmingen, San Diego, CA) at a dose of 2 μg/mL in a 1% bovine serum albumin in PBS overnight at 4°C. The plates are then washed, and culture supernatants are added and incubated overnight at 4°C. The plates are again washed and incubated with biotinylated antimouse IL-10 MoAb

(Pharmingen) at a dose of 2 μg/mL for 45 mins at room temperature. They are again washed and 100 μl of a 1:1000 dilution of peroxidase-streptavidine (Kirkegaard & Perry, Gaithersburg, MD) is added to each well, and incubated at room temperature for 30 mins. The plates are washed and lOOμl of freshly prepared 2,2' -azino-di (3-ethylbenzthiazoline-6- sulfonate) peroxidase solution (Kirkegaard & Perry) is added to each well. The reaction proceeds for 60 mins and is stopped by adding lOOμl of 1% SDS in PBS. Plate optical density is measured at 405 nm. A standard curve with purified IL-10 is performed for each assay. Example 5

Efficient gene expression from the HIV LTR is dependent on the viral protein tat. Tat mediates this effect by interacting with the TAR element in the HIV-1 LTR and can be taken up by cells in tissue culture to trans-activate the HIV-1 promoter. Because expression is dependent on tat, the HIV-1 promoter provides a way to introduce the SV40 (ts) to hepatocytes where it is expressed only when induced.

The viral protein rev also facilitates expression of HIV-1 genes. This protein is localized in the nucleus of HIV-1 infected cells and works downstream from tat to help transport RNA from the nucleus to the cytoplasm. The rev proteins binds to a complex stem loop structure, the rev response element (RRE), within the coding region of the HIV-1 env gene. In the absence of rev protein, only small multiply spliced RNAs accumulate in the cytoplasm. In the presence of rev, unspliced and singly spliced viral messenger RNAs can accumulate in the cytoplasm. By constructing a vector in which gene expression is dependent on tat and rev, it should be possible to introduce the SV40(ts) into primary hepatocytes so that the oncogene is not expressed until induced by both of these viral proteins.

A vector of this type has been constructed. This construct was made from the plasmid, polRRE (a gift from Dr. Stephen P. Goff, Dept. of Biochemistry, Columbia U.) , which contains the HIV LTR 5' to a cassette of genes including (5' to 3') the chloramphenicol acetyl transferase (CAT) gene, the HIV-1 polymerase gene, the RRE, and the SV40 poly A region, on a bluescript backbone. The CAT gene was excised from polRRE using the restriction enzymes Hindlll and Sail. The early region genomic fragment from SV40tsA58, encoding nucleotides 5235 to 2666, was excised from pZipSVtsA58 (Jat et al . SUPRA) (a gift from P.S. Jat, Ludwig Institute for Cancer Research, London) using BamHI and inserted into the CAT site using BamHI adapters. The resulting construct pSVpolRRE was then digested with Sail and Bglll to excise the polymerase gene and RRE and re-ligated using an adapter. The vector, termed pHIV-SVts, containing the HIV-1 promoter, the gene encoding the SV40 (ts) oncogene, and the SV40 poly A tail was used in preliminary studies to conditionally immortalize rat hepatocytes using the tat protein to transactivate the HIV-1 promoter. Since transactivation by tat is dramatically increased in the presence of chloroquine (which probably protects tat from proteolytic degradation) , a dose response curve using chloroquine is performed in order to determine the minimum concentration of tat necessary to transactivate the HIV-1 promoter and produce cell transformation by the SV40(ts) oncogene. A vector which uses the lac operon and the HIV-1 promoter to regulate oncogene expression is also being constructed. A commercially available system for inducible expression of introduced genes in eukaryotic cells (Lac Switch Inducible Mammalian Expression System, Stratagene, La Jolla, CA) may be used and consists of a eukaryotic lac-repressor-expressing vector, p3'SS, and a eukaryotic lac-operator-containing vector p0P13CAT into which the HIV-1 promoter and the SVtsA58 fragment can be inserted. These vectors are then transfected together into primary hepatocytes. Clones positive for both vectors are designed to be selected by their ability to grow in media containing hygromycin and G418. This is not necessary because SV40 (ts) expression and cell transformation can only take place when IPTG and tat protein are added to the culture media. When removed from the media, the oncogene is down-regulated, and the phenotype of the transduced cells return to normal. Primary rat and human hepatocytes are transfected with these plasmids.

In order to increase the efficiency of stable transduction and immortalization of non-rodent hepatocytes, we have successfully used recombinant adenoviruses which contain the SV40 oncogene. Persistent oncogene expression has been maintained in hepatocytes transformed by recombinant adenoviruses despite the fact that the viral DNA integrates into chromosomal DNA with limited efficiency. In order to create large numbers of human lines with tight oncogene regulation, a recombinant adenovirus may be constructed which utilizes the HIV-1 promoter and/or the RRE. This can be accomplished by using the 293 cell line, a human embryonic kidney cell line which constitutively provides the adenovirus-5 El gene products. Transfection of this cell line with pJM17 and pAC.RR25 (both provided by Dr. Robert Girard, Div. of Cardiology, U. of Texas Southwestern) , which contains a polycloning site allowing insertion of the HIV-1 promoter and the SV40(ts), results in the production, by homologous recombination, of a replication-defective recombinant adenoviruses containing these genes. Insertion of the polymerase gene, the RRE, and the poly A tail requires the use of recombinant adenovirus DL324 (provided by Dr. Thomas Shenk, Dept. of Molecular Biology, Princeton U.) instead of pJM17. This replication-defective virus can be used to create recombinant adenoviruses which contain inserts larger than 4.5 kb.

An alternative approach to protecting the recipient of conditionally immortalized cellular transplants from tumor formation would be to introduce "suicide" genes into the cells prior to transplantation. Cells modified to contain the HSVtk gene become sensitive to treatment with the antiviral agent gancyclovir (GCV) , whereas normal cells are unaffected by this agent. This gene is commonly used in molecular biology for negative selection in homologous recombination experiments and in the production of monoclonal antibodies. Gancyclovir is converted by HSVtk into nucleotide-like precursors that kill cells containing the gene by blocking DNA synthesis. Gancyclovir, which does not interact with human or rodent thymidine kinase, is not toxic to most tissues lacking HSVtk. Therefore, clones may be transfected with the HSVtk gene prior to being used for transplant experiments so that they can be destroyed post-transplantation by treatment with gancyclovir if recipients develop tumors. Example 6

Despite growing successes associated with whole organ liver transplantation, surgical risks and complications contribute significantly to patient morbidity and mortality. Hepatocyte transplantation could be used to treat acute liver failure and liver-based metabolic diseases and would avoid surgical intervention and its associated risks. A potential alternative to the transplantation of primary hepatocytes would be the use of a clonal cell line. A hepatocyte cell line provides the advantage of availability, uniformity and sterility and can be grown in unlimited quantity and at far less cost compared to isolated primary hepatocytes. Conditionally immortalized hepatocyte cell lines can be engineered to treat liver-based metabolic diseases and liver failure, to be non-tumorigenic and to circumvent rejection following transplantation in allogeneic recipients. Primary rat hepatocytes are conditionally immortalized using a virus vector that contains the gene encoding a thermolabile mutant of the SV40 large T antigen. These cells are characterized at the oncogene permissive and non-permissive temperatures (33° and 37°C, respectively) to determine their level of differentiated function. In order to more tightly regulate the expression of the oncogene, primary hepatocytes are transduced with plasmid or recombinant virus vectors that use multiple regulatory elements to control the expression of the SV40 temperature-sensitive oncogene.

Following in vitro characterization, cell lines are transplanted into Nagase analbuminemic rats and rats with experimentally induced liver failure to examine the ability of these cells to correct deficiencies in liver function in vivo. Finally, conditionally immortalized cell lines are genetically altered to express no major histocompatibility complex (MHC) Class I or Class II surface antigens and to locally elaborate the immunosuppressive cytokine IL-10. In summary, these studies determine whether conditionally immortalized hepatocyte cell lines can be engineered to correct metabolic and global liver deficiencies, be non-tumorigenic, immunologically inert, and safe for future clinical application. Methods: Lewis rat hepatocytes were transduced with a replication-defective recombinant retrovirus containing the gene encoding a thermolabile mutant of the SV40 T antigen.

Transformed hepatocytes (SVTS-hep) were subcloned and characterized at the permissive (33°C) and non-permissive

(39°/37°) temperatures. Lewis rats underwent end-to-side portocaval shunts and were subjected to ammonium acetate (AA) administration (3.4 mmol, i.p.) in order to produce a model of inducible hepatic encephalopathy.

Results: At 33°C, the SVTS-hep exhibited a transformed phenotype, synthesized DNA and doubles in number every 2-3 days. When cultured at 39°/37°C DNA synthesis and cell growth declined dramatically and clones demonstrated morphologic characteristics of differentiated hepatocytes. Encephalopathy was assessed in 3 groups of rats: Gl (n=7) portocaval shunt

(PCS) ; G2 (n=7) portocaval shunt and intrasplenic transplantation of 3xl0⁷ SVTS-hep 2 weeks post shunt surgery

(Pcs/HTx) ; G3 (n=7) sham-operation i.e. no PCS or HTx (SO) .

Evaluation of consciousness and reflex activity by means of a coma scale (Max: 15 points) and plasma ammonia measurements were performed prior to and after AA-administration. Plasma ammonia levels taken 30 min after AA-administration showed significant differences between groups 1 week post HTx

(Gl:1036±208.9 G2:686±174 G3:215±120 mmol/l) (p<0.05) .

Behavioral testing at the same time after AA-administration supported the biochemical findings (Gl:4.3+1.0 G2:13.0±2.5 G3:15.0±0.0 points) (p<0.005) . At 4 weeks post HTx ammonia levels continued to be different after AA-administration (Gl:1250±37 G2:632±245 G3:380±179 mmol/l) (p<0.05) and none of the G2-rats showed impairment of consciousness or reflex activity after being subjected to AA-administration (Gl:3.0±1.7 G2:15.0±0.0 G3:15.0±0.0 points) (p<0.005).

Conclusions: These data indicate that transplanted SVTS-hep can correct symptoms of hepatic encephalopathy and with further engineering might be of therapeutic use. Example 7

Viral vectors and protein carriers utilizing asialoglycoprotein receptor (ASGPR)-mediated endocytosis useful to transfer genes for the correction of bilirubin-UDP- glucuronosyltransferase (B-UGT) deficiency have been designed. Primary Gunn rat hepatocytes were immortalized by transduction with a recombinant Moloney murine leukemia virus expressing a thermolabile mutant SV40 large T antigen (tsA58) . Cell colonies that emerged after culturing at the permissive temperature (33°C) for 3 weeks were cloned.

At 33°, the immortalized hepatocyte clones exhibited a transformed phenotype, synthesized DNA and doubled in number every 2-3 days. Immunotransblot studies showed that when cultured at 33°C, these cells contained differentiated hepatocyte markers, including albumin, ASGPR and androsterone- UGT at approximately 5-10% of the level found in primary hepatocytes maintained in culture for 24 hrs. Glutathione-S- transferase Y_p (GST-Y_p) , an oncofetal protein, which is undetectable in primary hepatocytes, was expressed in these cells.

In contrast, when cultured at 39°C, DNA synthesis and cell growth stopped, and morphologic characteristics of differentiated hepatocytes were demonstrated by light and electron microscopy. Expression of albumin, ASGPR and androsterone-UGT increased to 25-40% of the level in primary hepatocytes, whereas GST-Y_p concentration decreased. RNA blot hybridization showed that albumin mRNA levels paralleled albumin concentrations. ASGPR function was demonstrated by internalization of Texas red-labelled asialoorosomucoid, and binding and degradation of ¹²⁵I-asialoorosomucoid. Glucuronidation of bilirubin by hepatic bilirubin-UDP- glucuronosyltransferase (B-UGT) is essential for effective excretion of bilirubin. Deficiency of B-UGT activity, inherited as an autosomal recessive trait, leads to Crigler- Najjar syndrome, Type I in man. This syndrome is characterized by life-long unconjugated hyperbilirubinemia and results in bilirubin-encephalopathy in infancy or adolescence. Homozygous Gunn rats have a similar metabolic defect and are an animal model for this syndrome. Therapeutic genes can be transferred into liver cells using recombinant retroviruses or asialoglycoprotein receptor

(ASGR) -mediated endocytosis. Cultures B-UGT-deficient primary

Gunn rat hepatocytes are difficult to use for this purpose because gene expression in these cells rapidly decreases. Transformed cells cannot be used either because they often lack the critical characteristics of differentiated hepatocytes, such as ASGPR, which are necessary for receptor-mediated gene targeting. A cell line was developed which lacks bilirubin-UGT but expresses a differentiated hepatocyte phenotype. A heat labile mutant SV40 T-antigen, which is degraded at 39°C has been used to conditionally immortalize hepatocytes. A recombinant retrovirus capable of transducing mammalian cells at high efficiency has been constructed recently and used to immortalize a variety of cell types. Recombinant retrovirus have been used to transduce primary Gunn rat hepatocytes as described herein. The immortalized hepatocytes proliferate at the permissive temperature (33°C) , but stop growing and express the characteristics of differentiated hepatocytes at 39°C. The conditionally immortalized B-UGT-deficient hepatocytes express functional ASGPR and are well suited for the evaluation of gene therapy vectors to correct B-UGT deficiency.

MATERIALS AND METHOD Animals : Inbred Gunn rats (150 g, male) were obtained from a colony derived from breeders provided by Dr. Carl Hansen of the National Institutes of Health (Bethesda, MD) and maintained in the Special Animal Core of the Marion Bessin Liver Research Center at the Albert Einstein College of Medicine. The rats were maintained on standard laboratory rat chow on a 12 hr light/dark cycle. All experiments were performed according to the humane guidelines provided by the animal research Institutional Review Board of the Albert Einstein College of Medicine.

Recombinant retrovirus expressing a thermolabile mutant SV40 large T antigen encoded by the early region mutant tsA58 : A psi-2 derived producer cell line for a recombinant retrovirus containing the genes encoding a temperature-sensitive SV40 large T antigen (tsA58) and neomycin phosphotransferase (Neo¹) was kindly provided by Dr. P.S. Jat of the Ludwig Institute for Cancer Research, London, U.K. This producer line provides a viral titer of 5 x IO⁴ neomycin resistant CFU/ml when assayed on NIH 3T3 cells. Isolation, immortalization and cloning of Gunn rat hepatocytes: Hepatocytes were isolated by in si tu collagenase perfusion of livers from Gunn rats. Viability of the isolated hepatocytes, as determined by trypan blue exclusion, was 90%. The cells were suspended in DME (Grand Island Biological Company, NY) containing 4% fetal calf serum, 0.2 μM Dexamethasone (Sigma), penicillin and streptomycin, and plated on Primaria tissue culture flasks (T75) at 4 x IO⁶ cells per flask. Cells were incubated at 37°C in a 95% air/5% C0₂ atmosphere for 48 hrs with a daily change of medium. Supernatant, containing the recombinant retrovirus, was harvested from confluent plates of producer cells 18 hrs after the addition of fresh medium and filtered through 0.45 μM filters. Hepatocytes were infected 48 hrs after establishment with 3 ml of viral stock per flask in the presence of 12 μg of polybrene (Aldrich Chemical Co., Milwaukee, WI) at 37°C for 4 hrs. The virus-containing media was then aspirated and cultures were maintained in DME containing 4% fetal calf serum, 0.2 μM dexamethasone, penicillin and streptomycin at 33°C. Seven days after retroviral infection, neomycin resistant hepatocytes were selected by using the neomycin analogue G418 (Grand Island Biological Company, New York) at 400 μg/ml. G418-resistant colonies emerged in 3 weeks; individual colonies were isolated using cloning rings, released by trypsinization, and expanded by culturing at 33°C.

Ini tial clone section based on expression of differentiation markers : Immortalization cell clones were screened for proteins that are preferentially expressed by differentiated hepatocytes using immunotransblot studies. Immunotransblot studies were performed using specific antibodies against three hepatocyte-specific rat proteins: serum albumin, ASGPR, and androsterone-UGT. Out of 10 immortalized clones, the two that contained the highest level of these three proteins were chosen for further characterization. Since preliminary experiments showed that ASGPR was expressed at a higher level in cells incubated at 37°C than at 39°C, cells were incubated at 39°C for 2 hrs to degrade the ^tsT-antigen, and then maintained at 37°C for 12 hrs before immunotransblot analysis in subsequent studies. In addition to the hepatocyte-specific markers, colonies were analyzed for the oncofetal market, glutathione S-transferase, isoform Y_p (GST-Y_p) . Northern blot hybridization: Total cellular RNA was extracted, resolved by electrophoresis 1% agarose gels and blotted on nitrocellulose membranes (Schleicher and Schuell) . The blots were hybridized with cDNA probes for rat serum albumin, ASGPR, androsterone-UGT, GST-Y_p, and _/S-actin. Cell morphology: Selected hepatocyte clones, maintained at 33°C or incubated at 39°C, as described above, were examined by light microscopy and transmission electron microscopy. Cytochemical staining for catalase was performed in order to visualize peroxisomes. ³H-Thymidine uptake : Immortalized hepatocyte clones were cultured for 24 hrs or 48 hrs on Primeria plastic tissue culture plates (Becton Dickinson Labware, Lincoln Park, New

Jersey) at 33°C or 39°C at densities of 3 x 10^s cells per plate

(35 mm diameter) . Cells were incubated with media containing 150 pmol of ³H-thymidine (20 Ci/mmol) at 37°C for 60 min. ³H- thymidine incorporation by primary hepatocytes cultured at 37°C was studied under the same conditions for comparison. The labeling medium was removed, the cells were washed with PBS and released using a non-enzymatic cell dissociation solution (Sigma, St. Louis, MO) . Aliquots of cells were used for the measurement of trichloroacetic acid precipitable radioactivity by scintillation counting using Hydroflour (National Diagnostics, Manville, NJ) . DNA was measured by a sensitive fluorimetric assay.

Determination of cell number: Cloned immortalized hepatocytes were plated at a density of 2 x IO⁶ cells per plate (100 mm diameter) and incubated at 33°C or 39°C for up to 72 hrs. At 24 hr intervals, cells were released with a cell dissociation solution described above and counted using a Coulter cell counter. Immunotransblot studies : Immortalized hepatocytes were grown to 75% confluence at 33°C. One group was maintained at 33°C for another 20 hrs while a second group was transferred to 39°C for 4 hrs to degrade the intracellular mutant T-antigen, and then cultured at 37°C for 16 hrs. Cells were released using the non-enzymatic cell dissociation solution, washed with 0.25 M sucrose in 20 mM Tris-HCl, pH 7.4 containing 1 mM EDTA and collected after centrifugation. Cells were then resuspended in the wash buffer at approximately 10 mg protein/ml and homogenized in a glass/teflon homogenizer. Protein concentration was determined in each aliquot. Homogenates, containing 100 μg protein were subjected to sodium dodecylsulfate/10% polyacrylamide gel electrophoresis and electroblotted to polyvinylidene difluoride membranes

(Millipore Corp., Bedford, MA) . Immunotransblot studies were performed using antibodies against rat serum albumin, rat ASGPR, rat androsterone-UGT and rat GST-Y_p.

Evaluation of ASGPR function : ASGPR-directed internalization Texas red-labeled asialoglycoprotein was performed as follows: Asialoorosomucoid (ASO) (Sigma, St. Louis, MO) was conjugated with Texas red according to the manufacturer's instructions. Hepatocytes were plated and grown on glass cove slips at 33°C until approximately 60% of the cover slip surface was covered. At this point, the cover slips were divided into two sets. A and B, and each set was divided into four groups (group 1 through group 4) . Set A was kept at 33°C, while Set B was cultured at 39°C for 2 hrs and then at 37°C for 16 hrs. After this, the culture media were replaced by serum-free DME medium and both sets were transferred to 4°C. For groups 1 and 3, Texas red-labeled ASO was added to the culture medium at 1 μg/ml. For groups 2 and 4, Texas red-labeled ASO and unlabeled ASO was allowed to attach to receptors by incubation for two hrs at 4°C, after which the unbound ASO was removed by extensive washing. To analyze internalization of the receptor- ligand complex, groups 3 and 4 were transferred to 37°C for 10 min. All samples were then washed with PBS and fixed in 3.5% paraformaldehyde in PBS at 4°C overnight. The fixed cells were then washed with 20 mM Tris-HCl, pH 7.4 containing 150 mM NaCI and 2 mM calcium, and mounted on glass slides with fluorescence mounting medium (Sigma, St. Louis, MO) .

To further assess functional endocytosis via the ASGPR in immortalized hepatocytes, degradation of ASO following receptor-mediated endocytosis was evaluated. In brief, cells were grown at 33°C to approximately 2 x IO⁶ cells per 65 mm diameter plate. One group was incubated at the permissive temperature (33°C) for 20 hrs while a second group was incubated at 39°C for 2 hrs before culturing at 37°C for 16 hrs. Duplicate plates were then incubated with ¹²⁵I-ASO, for 30 min at 4°C for surface binding without internalization. Unbound ASO was removed by washing in ice-cold DME containing 2 mM CaCl₂. From one set of plates, cells were released by scraping with a rubber policeman, and bound radioactivity was determined in a gamma counter. To the other set of plates, 1 ml of culture medium (DME with 4% fetal calf serum) was added and the cells were incubated at 37°C for 2 hrs. After this incubation, cells were precipitated in 10% ice-cold TCA, pelleted by centrifugation, washed with ice-cold 10% TCA and assessed for radioactivity. Transplantation into immunodeficient mice: To test whether the immortalized hepatocytes are tumorigenic, the cells were transplanted into two immunodeficient SCID mice. For comparison, cells of a differentiated human hepatoma line (HepG2) were transplanted into two other SCID mice. For comparison, cells of a differentiated human hepatoma line (HepG2) were transplanted into two other SCID mice. Each SCID mouse was injected 2 x IO⁶ immortalized rat hepatocytes or HepG2 cells into the splenic pulp and 2 x IO⁶ cells in the subcutaneous fat pad of the right flank. After 4 weeks the mice were killed. Subcutaneous tissue at the site of injection and the spleens were resected and frozen sections were examined after staining with hematoxylin and eosin and for glucose-6- phosphatase activity.

RESULTS Cell Morphology: Light microscopy of the cultured immortalized cells was performed using phase contrast. Cells grew in monolayers and demonstrated intercellular junctions. Cells at the center of each colony were smaller, more elongated and had greater cytoplasm to nucleus ratios, compared to those situated at the periphery of each colony. Cells cultured at 33°C had a greater relative proportion of the smaller central cells than did cells maintained at 39°C or 37°C for 16 hrs or longer. Cells were cultured at 33°C or 39°C and stained for catalase activity to visualize peroxisomes. Large peroxisomes, which are characteristic of differentiated hepatocytes, were observed. Transmission electron microscopy showed intercellular junctions with microvilli characteristic of bile canaliculi.

DNA synthesis : ³H-thymidine incorporation by immortalized cells cultured at 33°C or 39°C was compared. The incorporation of ³H-thymidine by the immortalized cells cultured for 24 hrs or 48 hrs at the permissive temperature (33°C) was 25 to 50 fold greater than that by primary hepatocytes cultured at 37°C. When cultured at the non-permissive temperature (39°C) for 24 hrs, DΝA ³H-thymidine incorporation was reduced to approximately 2-fold that of primary hepatocytes. When the cells were cultured at 39°C for 48 hrs, DΝA synthesis rate in the immortalized cells approximately equalled that in primary hepatocytes. Cell proliferation : At 33°C, the immortalized cells doubled in number in approximately 72 hrs. There was no significant increase in cell number when the immortalized cells were cultured at 39°C or when primary hepatocytes were cultured at 33°C or 39°C.

Expression of hepatocytes marker proteins : To measure protein expression or cellular function, cells were maintained at 33°C or at 37°C after an initial incubation for 4 hrs at 39°C to degrade the ^tsT-antigen. The rationale for maintaining cells at 37°C was that in preliminary experiments, ASGPR was expressed at 37°C at a much higher level than at 39°C. Immunotransblot studies showed that immortalized cells cultured at 33°C contained albumin, ASGPR and androsterone-UGT at approximately 5-10% of the levels found in primary hepatocytes cultured for 24 hrs under the same conditions. In contrast, GST-Y_p, an oncofetal protein, was expressed at a 10-times higher level. When cultured at 37°C, cellular concentrations of albumin, ASGPR and androsterone-UGT increased to 25-40% of the level in primary hepatocytes while GST-Y_p concentration decreased markedly.

Northern blots : Northern blot analysis with probes specific for albumin and androsterone-UGT showed that the cellular concentration of these mRNAs paralleled the changes in the concentration of the corresponding proteins. Concentrations of _/8-actin mRNA and ribosomal RNA subunits remained constant at the various culture conditions.

ASGPR function : The function of ASGPR in conditionally immortalized hepatocytes was evaluated in the following three experiments. (a) Uptake of Texas -red-labeled asialoorosomucoid

(ASOR) : Internalization of ASOR was demonstrated by the finding of fluorescence-labeled ASOR within the cultured cells. Cells cultured at 37°C showed internalization of a large number of fluorescent vesicles, which was mostly abolished when the uptake experiment was performed in the presence of excess unlabeled ASOR. Cells cultured at 33°C demonstrate a greatly reduced number of fluorescent vesicles compared to those cultured at 37°C.

(b) Binding and degrada tion of ¹²⁵l- labeled ASOR : Binding of radiolabeled ASOR by cells cultured at 33°C was approximately 30% of that by cultured primary hepatocytes. At 39°C, the binding increased to approximately 55% of the level in primary hepatocytes. Under both culture conditions, surface binding was reduced by 90-98% when the experiment was performed in the presence of excess unlabeled ASOR. Internalization and degradation of ASOR was determined by the appearance of acid soluble breakdown products of the bound ASOR into the medium after incubation at 37°C for 2 hrs. Cells maintained at 33°C degraded ASOR at approximately 23% of the rate observed in primary hepatocytes. In cells cultured at 37°C, the rate of ASOR degradation was approximately 54% that in primary hepatocytes.

Transplanta tion into SCID mice : Four weeks after transplantation, the recipients of HepG2 cells developed large tumors (1-2 cm diameter) at the site of subcutaneous injection. Their livers and spleens were studded with tan colored tumors of various sizes. These rats lost approximately 50% of their body weight. In contrast, recipients of the immortalized hepatocytes exhibited no visible tumors in the subcutaneous tissue, spleen or liver. Histological examination of spleens of these mice showed the presence of cells that had the morphology of hepatocytes and were positive for glucose-6- phosphatase activity.

DISCUSSION We have pursued two approaches for liver directed gene therapy in the treatment of inherited metabolic abnormalities of the liver. In ex vivo gene therapy, hepatocytes are isolated from metabolically deficient host and are established in primary culture. Normal therapeutic genes are then introduced into these hepatocytes by transduction with recombinant retroviruses. After phenotypic correction, the transduced hepatocytes are transplanted back into the host. Protein carriers that utilize asialoglycoprotein receptor (ASGPR) -mediated endocytosis can also be used.

Cells are infected with a virus that expresses a transforming gene containing a temperature-sensitive mutation. At the permissive temperature, the transforming gene product produces unlimited growth, along with loss of differentiated function. At the higher (non-permissive) temperature, the cells have restricted growth potential and express differentiated cell functions. Such an SV40 virus, containing a temperature-sensitive mutation in the gene encoding the large T nuclear antigen, has been used to derive conditionally immortalized hepatocytes. To increase the efficiency of transformation, a recombinant retrovirus, expressing the thermolabile SV40 large T antigen has been constructed. This recombinant retrovirus to obtain conditional immortalization of Gunn rat hepatocytes.

Although 39°C is the optimum temperature for degradation of the thermolabile T antigen, expression of many differentiated hepatocyte proteins is reduced at this temperature compared to that at 37°C. For this reason, we first incubated the cells for 4 hrs at 39°C to degrade the T antigen before transferring them to 37°C to study differentiated hepatocyte functions.

Expression of the Y_p isoform of the GST family increases in conditions associated with rapid hepatocellular proliferation, such as carcinogen-induced hepatic preneoplastic nodules. Relative abundance of mRNA for this isoform increases when primary hepatocytes are cultured for longer than 24 hrs. Under these conditions, expression of this protein is inversely proportional to the expression of proteins specifically expressed by differentiated hepatocytes, such as albumin and GST-Y_a. Interestingly, this inverse relationship between the expression of GST-Y_p and hepatocyte-specific proteins was also observed in the immortalized hepatocytes cultured at permissive versus non-permissive temperatures.

Retention of hepatocellular morphology and continued expression of glucose-6-phosphatase activity by the immortalized hepatocytes transplanted in the spleen of SCID mice indicate their potential for surviving and function in vivo . During the 4 weeks following transplantation, HepG2 cells, a relatively slow-growing cell line, developed large tumors in the recipient mice. In contrast, the immortalized cells showed no sign of tumorigenesis during this period. Longer germ observation is needed to determine whether, with appropriate safe-guards, conditionally immortalized hepatocytes can be transplanted without the risk of tumorigenesis.

Claims

1. A recombinant hepatocyte cell that comprise: a recombinant expression cassette which contains a nucleotide sequence that encodes a condition sensitive transforming protein operable linked to at least one activatable regulatory element; and a gene that encodes a protein which can be targeted for selective elimination.

2. The recombinant hepatocyte of claim 1 wherein said recombinant expression cassette comprises three activatable regulatory elements.

3. The recombinant hepatocyte of claim 2 wherein said activatable regulatory element are an HIV 5' LTR, an HIV RRE and a lac operon.

4. The recombinant hepatocyte of claim 3 wherein said nucleotide sequence that encodes a condition sensitive transforming protein encodes a temperature sensitive SV40 T antigen.

5. The recombinant hepatocyte of claim 5 wherein said gene that encodes a protein which can be targeted for selective elimination is a Herpes Simplex Virus thymidine kinase gene.

6. The recombinant hepatocyte of claim 1 wherein said nucleotide sequence that encodes a condition sensitive transforming protein encodes a temperature sensitive SV40 T antigen.

7. The recombinant hepatocyte of claim 1 wherein said activatable regulatory element is a selected from the group consisting of: a metallotheonein promoter, an HIV 5' LTR, an HIV RRE and a lac operon.

8. The recombinant hepatocyte of claim 1 wherein said gene that encodes a protein which can be targeted for selective elimination is a Herpes Simplex Virus thymidine kinase gene.

9. The recombinant hepatocyte of claim 1 comprising three activatable regulatory elements wherein said nucleotide sequence that encodes a condition sensitive transforming protein encodes a temperature sensitive SV40 T antigen; said three activatable regulatory elements are selected from the group consisting of: a metallotheonein promoter, an Human Immunodeficiency virus 5' Long Terminal Repeat, an HIV RRE and an E. coli lac operon; and said gene that encodes a protein which can be targeted for selective elimination is a Herpes Simplex Virus thymidine kinase gene.

10. The recombinant hepatocyte of claim 1 comprising three activatable regulatory elements wherein said nucleotide sequence that encodes a condition sensitive transforming protein encodes a temperature sensitive SV40 T antigen; said three activatable regulatory elements are: an Human Immunodeficiency virus 5' Long Terminal Repeat, an HIV RRE and an E. coli lac operon; and said gene that encodes a protein which can be targeted for selective elimination is a Herpes Simplex Virus thymidine kinase gene.

11. The recombinant hepatocyte of claim 10 wherein said cell comprises a third exogenous gene.

12. The recombinant hepatocyte of claim 1 wherein said cell is modified to reduce or eliminate production of major histocompatibility proteins.

13. The recombinant hepatocyte of claim 1 wherein said cell comprises a third exogenous gene.

14. The recombinant hepatocyte of claim 1 wherein said cell is microencapsulated.

15. An implantable device comprising a plurality of cells according to claim 1.

16. A method of treating an individual suspected of suffering from a liver insufficiency condition comprising the step of: introducing into an individual suspected of suffering from a liver insufficiency condition, a plurality of recombinant hepatocyte cells of claim 1.

17. The method of claim 16 wherein said nucleotide sequence that encodes a condition sensitive transforming protein encodes a temperature sensitive SV40 T antigen.

18. The method of claim 16 wherein said gene that encodes a protein which can be targeted for selective elimination is a Herpes Simplex Virus thymidine kinase gene.

19. The method of claim 16 wherein said activatable regulatory element is a selected from the group consisting of: a metallotheonein promoter, an HIV 5' LTR, an HIV RRE and a lac operon.

20. The method of claim 16 wherein said gene that encodes a protein which can be targeted for selective elimination is a Herpes Simplex Virus thymidine kinase gene.

21. The method of claim 16 comprising three activatable regulatory elements wherein said nucleotide sequence that encodes a condition sensitive transforming protein encodes a temperature sensitive SV40 T antigen; said three activatable regulatory elements are: an Human Immunodeficiency virus 5' Long Terminal Repeat, an HIV RRE and an E. coli lac operon; and said gene that encodes a protein which can be targeted for selective elimination is a Herpes Simplex Virus thymidine kinase gene.

22. The method of claim 16 wherein said cell is modified to reduce or eliminate production of major histocompatibility proteins.

23. The method of claim 16 wherein said cell comprises a third exogenous gene.

24. The method of claim 16 wherein said cell is microencapsulated.

25. The method of claim 16 wherein said cells are in an implantable device.