Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1995-06-06
2001-11-13
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S370000, C424S093210
Reexamination Certificate
active
06316255
ABSTRACT:
FUNDING
Work described herein was funded by the Howard Hughes Medical Institute, the National Institutes for Health and the Whitehead Institute for Biomedical Research.
BACKGROUND
The liver is of endodermal origin and the largest gland in the human body. It has numerous crucial roles, including bile secretion, participation in carbohydrate, lipid and protein metabolism, fibrinogen production and detoxification of drugs. The liver also serves as the main site at which nutrients absorbed from the gastrointestinal tract and transported via the blood are processed for use by other body cells.
Hepatocytes, which are the main type of parenchymal or distinguishing cell in the liver, carry out the liver functions and, thus, are responsible for synthesizing, degrading and storing a wide variety of substances. In addition, a system of small channels (canaliculi) and larger ducts connects hepatocytes with the gut lumen. Through this route, hepatocytes secrete bile, an emulsifying agent which helps in absorption of ingested fats. Hepatocytes are also the main location at which lipoprotein particles for export are made; enzymes responsible for synthesis of the lipid constituents of lipoproteins occur in hepatocyte membranes.
Because of the many important functions the liver has, its inability to function normally (e.g., as a result of a genetic defect or damage caused by alcohol or other toxic substances) will often have significant adverse effects on an individuals health. A means by which normal function can be conferred upon or restored to a liver whose function is compromised would be very useful in treating, correcting or preventing such an abnormality.
SUMMARY OF THE INVENTION
The invention described herein relates to genetically engineered or transduced hepatocytes which express genetic material (DNA or RNA) of interest introduced or incorporated into them, as well as to methods of producing, transplanting and using the genetically engineered hepatocytes. The genetic material of interest can be incorporated through the use of a vector, such as a recombinant retrovirus, which contains the genetic material of interest, or by other means.
Hepatocytes of the present invention express the genetic material of interest. Such genetic material of interest can be: 1) genetic material present in and expressed at biologically effective levels by normal hepatocytes, but present in or expressed in less than normal quantities in the hepatocytes prior to transfer of genetic material of interest into them by the method of the present invention; 2) genetic material not present in normal hepatocytes; or 3) genetic material present in normal hepatocytes but not expressed at biologically effective levels in such cells, alone or in any combination thereof.
In hepatocytes of the present invention, the genetic material of interest can be incorporated into the cellular genetic material (e.g., into genomic DNA) or can be present extrachromosomally (i.e., expressed episomally). The genetic material of interest can be DNA or RNA; the DNA can constitute all or a portion of a gene of interest (i.e., one whose expression in hepatocytes is desired).
The genetic material incorporated into and expressed by hepatocytes of the present invention can additionally include genetic material (e.g., DNA) encoding a selectable marker, which provides a means by which cells expressing the genetic material of interest are identified and selected for. Hepatocytes containing incorporated genetic material (i.e., genetic material of interest and, optionally, genetic material encoding a selectable marker) are referred to as transduced hepatocytes.
Genetic material can be introduced into hepatocytes ex vivo or in vivo. That is, it can be introduced, by means of an appropriate vector, into isolated (cultured) hepatocytes, which are subsequently transplanted into the recipient. Alternatively, it can be introduced directly into the recipient in such a manner that it is directed to-and taken up by, target cells (hepatocytes), where it is incorporated and expressed. Particularly useful for this purpose are retroviral vectors which have an amphotropic host range and include the genetic material of interest which is to be incorporated into hepatocytes.
Retroviral vectors have been used to stably transduce hepatocytes with genetic material which included genetic material encoding a polypeptide or protein of interest and genetic material encoding a dominant selectable marker. Genetic material including DNA encoding a polypeptide of interest and DNA encoding a dominant selectable marker has been introduced into cultured hepatocytes. Expression of the genetic material by the hepatocytes into which they have been incorporated has also been demonstrated.
A method of transplanting transduced hepatocytes which express the incorporated genetic material they contain is also a subject of the present invention. Transduced hepatocytes of the present invention are used, for example, for the delivery of polypeptides or proteins which are useful in prevention and therapy of an acquired or an inherited defect in hepatocyte (liver) function. For example, they can be used to correct an inherited deficiency of the low density lipoprotein receptor (LDLR), which is synthesized in hepatocytes, and to correct an inherited deficiency of ornithine transcarbalyase (OTC), which results in congenital hyperammonemia.
Hepatocytes of the present invention are useful as a means by which abnormal hepatocyte function can be corrected. That is, hepatocytes can be transduced with genetic material of interest selected to compensate for over- or underproduction of a protein or peptide which is synthesized correctly, but in inappropriate amounts in the hepatocytes. Alternatively, they can be transduced with genetic material of interest encoding a protein or polypeptide which is produced in an appropriate quantity, but is functionally defective (e.g., because of an abnormal structure or amino acid makeup).
Hepatocytes to be modified ex vivo, as described herein, can be obtained from an individual, modified and returned to the individual by transplanting or grafting or, alternatively, can be obtained from a donor (i.e., a source other than the ultimate recipient), modified and applied to a recipient, again by transplanting or grafting.
An important advantage of the procedure of the present invention is that the genetically engineered hepatocytes can be used to provide a desired therapeutic protein or peptide by a means essentially the same as that by which the protein or peptide is normally produced and, in the case of autologous grafting, with little risk of an immune response and graft rejection. In addition, there is no need for extensive (and often costly) purification of a polypeptide before it is administered to an individual, as is generally necessary with an isolated polypeptide. Hepatocytes modified according to the present invention produce the polypeptide as it would normally be produced.
Because genes can be introduced into hepatocytes using a retroviral vector, they can be subject to the retroviral vector control. In such a case, the gene of interest is transcribed from a retroviral promoter. A promoter is a specific nucleotide sequence recognized by RNA polymerase molecules that start RNA synthesis. Alternatively, retroviral vectors having additional promoter elements, which are responsible for the transcription of the genetic material of interest, can be used. For example, a construct in which there is an additional promoter modulated by an external factor or cue can be used, making it possible to control the level of polypeptide being produced by the modified hepatocytes by activating that external factor or cue. For example, heat shock proteins are proteins encoded by genes in which the promoter is regulated by temperature. The promoter of the gene which encodes the-metal-containing protein metallothionine is responsive to cadmium (Cd
++
) ions. Incorporation of this promoter or another promoter influenced by external cues also makes it possible to regula
Mulligan Richard C.
Wilson James M.
Crouch Deborah
Pennie & Edmonds LLP
Whitehead Institute for Biomedical Research
LandOfFree
Hepatocytes transduced with a retroviral vector comprising... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Hepatocytes transduced with a retroviral vector comprising..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Hepatocytes transduced with a retroviral vector comprising... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2573749