Retroviral gene transfer into diploid fibroblasts for gene therapy

• US 5,219,740 A
• Filed: 02/13/1987
• Issued: 06/15/1993
• Est. Priority Date: 02/13/1987
• Status: Expired due to Term

• Current Assignee: Fred Hutchinson Cancer Research Center (Fred Hutchinson Cancer Research Center|Argus Genetics|Mars)
• Original Assignee: Fred Hutchinson Cancer Research Center (Fred Hutchinson Cancer Research Center|Argus Genetics|Mars)
• Inventors: Palmer, Theo D., Miller, A. Dusty
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): RAILEY, JOHNNY F
• Application Number:
  US07/014,579
• Time in Patent Office: 2,314 Days
• Field of Search: 435/68, 435/172.3, 435/235, 435/240.1, 435/172.1, 435/236, 435/239, 435/240.2, 435/240.21, 435/320, 435/948, 424/89, 424/91, 424/93, 424/94, 424/95, 424/94.1, 935/6, 935/22, 935/32, 935/34, 935/57, 935/60, 935/62, 935/63, 935/66, 935/70, 935/71, 935/79
• US Class Current: 435/69.6
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 49/00 Preparations for testing in vivo
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2740/13043 viral genome or elements thereof as genetic vector

Abstract

A process of mammalian gene therapy. Explanted fibroblasts are genetically modified by introducing a retroviral construct containing a nucleotide sequence encoding for a therapeutic substance. The genetically modified fibroblasts are implanted into a mammalian subject.

Claims

1. A process of gene transfer into diploid fibroblasts in vitro, comprising the step of:

genetically modifying diploid fibroblasts explanted from a mammalian subject by a process consisting essentially of introducing into said fibroblasts a retroviral construct comprising a first nucleotide sequence encoding a first expression product, a viral long terminal repeat and a promoter sequence upstream of said first nucleotide sequence, and a viral long terminal repeat and a polyadenylation sequence downstream of said first nucleotide sequence, wherein said retroviral construct lacks one or more of the gag, pol, and env sequences required for retroviral replication, by contacting said fibroblasts with said retroviral construct in a virus-containing medium having a viral titer of at least about 10⁵ cfu/ml on NIH 3T3 fibroblasts to produce a population of fibroblasts at least 1% of which express said first expression product.

2. The process of claim 1, wherein said retroviral construct further comprises a second nucleotide sequence encoding a second expression product, and wherein one of the first and the second expression products confers resistance to a selectable marker substance.

3. The process of claim 2, wherein said selectable marker substance is selected from the group consisting of G418 and Hygromycin-B.

4. The process of claim 1, wherein said diploid fibroblasts are explanted from the tissue of a mammalian subject.

5. The process of claim 1, wherein said mammalian subject is a human.

6. The process of claim 2, wherein the other of the first and the second expression products is selected from the group consisting of enzymes, hormones, and precursors thereof.

7. The process of claim 6, wherein the other of the first and the second expression products is selected from the group consisting of adenosine deaminase, purine nucleoside phosphorylase, and blood clotting factors.