Plasmid for delivery of nucleic acids to cells and methods of use

• US 5,763,270 A
• Filed: 06/07/1995
• Issued: 06/09/1998
• Est. Priority Date: 06/07/1995
• Status: Expired due to Term

• Current Assignee: Valentis, Inc. (Vaneltix Pharma, Inc.)
• Original Assignee: GeneMedicine Co LTD
• Inventors: Eastman, Eric M., Durland, Ross H.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/484,723
• Time in Patent Office: 1,098 Days
• Field of Search: 435/320.1, 435/6, 435/5, 514/44, 536/23.1, 935/22, 935/23, 935/24, 935/29, 935/52, 935/55, 935/56, 935/66, 935/72
• US Class Current: 435/320.1
• 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: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2820/002 inducible or controllable
• CPC Class Codes: C12N 2820/55 from bacteria
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/006 tet repressible
• CPC Class Codes: C12N 2830/36 being a transcription termination element
• CPC Class Codes: C12N 2830/55 from bacteria
• CPC Class Codes: C12N 2840/20 translation of more than one cistron

Abstract

This invention relates to plasmid constructs useful for nonviral human gene therapy. The plasmid constructs incorporate plasmid elements for achieving high copy number, avoiding plasmid instability an providing a plasmid selection process. These constructs can be used to deliver nucleic acids to cells in vehicles which are compatible with human therapeutic use.

Claims

1. A plasmid for delivery of a nucleic acid sequence to a eukaryotic cell, comprising:

(a) a region which regulates plasmid copy number, wherein said region includes an origin of replication sequence;

(b) a region which regulates selection of a plasmid containing cell;

(c) a linker region connecting said region which regulates plasmid copy number to said region which regulates selection of a plasmid containing cell, wherein said linker region includes a site for inserting said nucleic acid sequence, and a transcriptional terminator element to ensure transcription of said region which regulates selection of a plasmid containing cell does not transcribe through said site for insertion of said nucleic acid sequence;

(d) a plurality of nucleic acid restriction sites flanking said origin of replication of said region which regulates plasmid copy number, said linker region, and said transcriptional terminator sequence; and

(e) a functional element inserted in a said nucleic acid restriction site, wherein said functional element is selected from the group consisting of a partitioning locus which helps ensure that at least one copy of said plasmid is transmitted to each new cell during cell division and a multimer resolution locus which promotes plasmid monomerization,

wherein said region which regulates plasmid copy number, said linker region, and said region which regulates selection of a plasmid containing cell are positioned 5'"'"' to 3'"'"' in the order;

region which regulates plasmid copy number, linker region, region which regulates selection of a plasmid containing cell, and wherein the transcriptional orientation of said region which regulates selection of a plasmid containing cell is opposite to the transcriptional orientation of a said nucleic acid sequence inserted in said linker region.

2. The plasmid of claim 1, wherein said region which regulates selection of a plasmid containing cell comprises a nucleic acid sequence encoding for antibiotic resistance.

3. The plasmid of claim 1, wherein said functional element is a partitioning locus.

4. The plasmid of claim 1, wherein said functional element is a multimer resolution locus.

5. The plasmid of claim 2, wherein

(a) said origin of replication is from the plasmid pMM1,

(b) said nucleic acid sequence encoding for antibiotic resistance comprises the neo gene,

(c) said multicloning site is the polylinker sequence from pBluescript KS+, and

(d) said transcriptional terminator sequence is rrnBT₁ T₂.

6. A system for selection of a cell containing a plasmid, wherein said system comprises:

(a) a plasmid of claim 1; and

(b) a cell containing at least one selection gene incorporated into a chromosome of said cell under the control of a chromosomal operon, wherein said operon is capable of derepression by an operator sequence present on said plasmid by repressor titration.

7. The system of claim 6, wherein said selection gene is an antibiotic resistance gene.

8. The system of claim 6, wherein said operator sequence on said plasmid does not specifically bind a repressor naturally found in said cell.

9. A method for delivery of a nucleic acid sequence to a cell, wherein said method comprises the step of contacting said cell with a plasmid of claim 1 for sufficient time to transform said cell,

wherein said plasmid further comprises a said nucleic acid sequence.

10. A method for selection of a host cell containing a plasmid, wherein said method comprises the steps of:

(a) selecting a plasmid which appropriately transforms a first cell by contacting said first cell with a plasmid for sufficient time to transform said cell,

wherein said plasmid is a plasmid of claim 1, further comprising an antibiotic resistance nucleic acid sequence and a site for insertion of a nucleic acid sequence;

(b) removing said antibiotic resistance nucleic acid sequence from said selected plasmid at flanking nucleic acid restriction sequences;

(c) recircularizing said selected plasmid to provide a selected plasmid lacking said antibiotic resistance nucleic acid sequence;

(d) contacting a second cell with said selected plasmid lacking said antibiotic resistance nucleic acid sequence for sufficient time to transform said second cell,

wherein said second cell contains at least one antibiotic resistance gene incorporated into a chromosome of said cell under the control of a chromosomal operon, wherein said operon is capable of derepression by the operator sequence present on said selected plasmid; and

(e) selecting a transformed second cell resistant to antibiotic.

11. A method for obtaining a selected plasmid lacking an antibiotic resistance nucleic acid sequence, comprising the step of isolating the selected plasmid lacking an antibiotic resistance nucleic acid sequence from the transformed second cell of claim 10.