# US 2014/0193914 A1 · Introduction of Modular Vector Elements During Production of a Lentivirus

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- Number: US 2014/0193914 A1
- Document ID: US20140193914A1
- Status: Published application
- Kind: A1
- Application number: 14/236,083
- Filed: 2012-09-13
- Published: 2014-07-10
- Earliest claimed: 2011-09-23
- Assignees: Thermo Fisher Scientific Biosciences Inc.
- Inventors: Jamie Cearley; Rusla DuBreuil
- Web page: https://patentgrep.com/patent/US20140193914A1

## CPC

- C12N 15/86: Viral vectors
- C12N 15/85: for animal cells
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
- C12N 2800/40: Systems of functionally co-operating vectors
- C12N 2800/50: Vectors for producing vectors
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods and kits for lentiviral production are provided. By separating certain components of the vector including but not limited to promoter, reporter and selection marker preference components from the genetic content of interest, one can create libraries that allow for the efficient generation of custom selected lentiviral vectors.

## Claims

### Claim 1

A kit for the introduction of modular vector elements comprising:
(a) a preference vector, wherein said preference vector comprises a promoter sequence; and
(b) a content vector, wherein said content vector comprises a genomic content of interest, a first LTR sequence that is 5′ of the genomic content of interest and a second LTR sequence that is 3′ of the genomic content of interest, and wherein said content vector does not comprise any promoter sequence and said preference vector does not comprise said genomic content of interest and further wherein said preference vector and said content vector each contain a single recombination site and are capable of combining through a cross-over event to form a lentiviral vector.

### Claim 2 (depends on 1)

The kit according to claim 1, wherein the preference vector further comprises a selection marker sequence and a reporter sequence.

### Claim 3 (depends on 2)

The kit according to claim 2, wherein the preference vector is selected from the group consisting of pGK-GFP, pCAGGS-LUC, SV40-CYN and pGK-RFP.

### Claim 4 (depends on 1)

The kit according to claim 1, further comprising a plurality of cells from an engineered packaging cell line.

### Claim 5 (depends on 1)

The kit according to claim 1, wherein the genomic content of interest content vector comprises a nucleotide sequence of an siRNA, an shRNA, an miRNA, a cDNA or an ORF.

### Claim 6 (depends on 1)

The kit according to claim 1, further comprising viral packaging plasmids.

### Claim 7

A kit for the introduction of modular vector elements comprising:
(a) a plurality of preference vectors, wherein said preference vectors each comprise a promoter sequence and a single recombination site;
(b) a plurality of cells from a packaging cell line; and
(c) at least one content vector, wherein each content vector comprises a sequence corresponding to a genomic content of interest, a single recombination site, a first LTR sequence that is 5′ of the genomic content of interest and a second LTR sequence that is 3′ of the genomic content of interest, and wherein each content vector has an absence of any promoter sequences, and each preference vector does not comprise any sequence that corresponds to said genomic content of interest, and further, wherein at least two preference vectors have different promoter sequences.

### Claim 8 (depends on 7)

The kit according to claim 7, wherein said kit comprises a plurality of content vectors, wherein the content vectors differ only by their genomic content of interest.

### Claim 9

A method for generating a viral vector comprising;
(a) introducing a preference vector, into a cell, wherein said preference vector contains a single recombination site and wherein the cell comprises one or more packaging plasmids;
(b) introducing a content vector into said cell, wherein said content vector contains a single recombination site, a sequence that corresponds to genomic content of interest, a first LTR sequence 5′ of the genomic content of interest and a second LTR sequence 3′ of the genomic content of interest; and
(c) subjecting said cell to conditions under which the preference vector and the content vector are capable of combining into a viral transcript, wherein said viral transcript is a lentiviral vector.

### Claim 10 (depends on 9)

The method according to claim 9, wherein the cell expresses Cre recombinase.

### Claim 11 (depends on 3)

The kit of claim 3, wherein the preference vector further comprises an On site and an EM7-Zeo site.

### Claim 12 (depends on 11)

The kit of claim 11, wherein the single recombination site of the content vector is Lox66 and the single recombination site of the preference vector is a Lox77 site.

### Claim 13 (depends on 12)

The kit of claim 12, wherein the Ori site of the preference vector is SV40-Ori and the content vector contains an SV40-Ori site.