Single lentiviral vector system for induced pluripotent (ips) stem cells derivation
AssigneeBoston Medical Center Corporation
InventorsGustavo Mostoslavsky, Cesar A. Sommer
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Abstract
The present invention is based on the discovery that a single lentiviral vector expressing multiple individual transcription factor proteins from a single multi-cistronic mRNA can reprogram a fibroblast cell to a stem cell-like cell. These reprogrammed induced pluripotent stem (iPS) cells are pluripotent. Additions of the Cre-LoxP sequences into the single lentiviral vector facilitate excision of the vector after reprogramming in achieved. Addition of a maker gene into the single lentiviral vector facilitates detection of the presence of the vector in an iPS. The invention provides compositions and methods of producing iPS cells using a single multi-cistronic lentiviral vector.
Claims22
A lentiviral vector particle wherein the vector particle comprises a nucleic acid sequence comprising a sequence encoding:
- a. a first gene;
- b. a second gene;
- c. a third gene;
- d. an optional fourth gene,
- wherein the first, second, third and optional fourth genes are selected from the group consisting of Oct4, Klf4, Sox2, cMyc, Lin28, and Nanog;
- and wherein the first, second, third and optional fourth genes are not identical;
- e. a first ‘self-cleaving’ 2A peptide;
- f. a second ‘self-cleaving’ 2A peptide;
- and
- g. an internal ribosome entry site (IRES);
- wherein the nucleic acid sequence is operably linked to a promoter,
- wherein the sequences encoding the first, second, third and optional fourth genes, first and second ‘self-cleaving’ 2A peptides, and the IRES are transcribed from the promoter as a multi-cistronic RNA.
A vector system comprising:
- a. a first vector containing a lentiviral gag gene encoding a lentiviral gag protein, wherein the lentiviral gag gene is operably linked to a promoter and a polyadenylation sequence,
- b. a second vector containing an env gene encoding a functional envelope protein, wherein the env gene is operably linked to a promoter and a polyadenylation sequence;
- c. a lentiviral pol gene encoding a lentiviral pol protein, wherein the pol protein is at least an integrase, and the pol gene is on the first or second vectors or on at least a third vector, wherein the lentiviral pol gene is operably linked to a promoter and a polyadenylation sequence;
- wherein the first, second and the at least third vectors do not contain sufficient nucleotides to encode the lentiviral gag and pol and the envelope protein on a single vector;
- and
- wherein the vectors do not contain nucleotides of the lentiviral genome referred to as a packaging segment to effectively package lentiviral RNA;
- and
- wherein the lentiviral proteins and the envelope protein when expressed in combination form a lentivirus virion containing an envelope protein around a lentiviral capsid;
- and
- d. a packaging gene transfer plasmid comprising a stem cell cassette nucleic acid sequence encoding:
- a first gene;
- a second gene;
- a third gene;
- an optional fourth gene;
- a first ‘self-cleaving’ 2A peptide;
- a second ‘self-cleaving’ 2A peptide;
- and an internal ribosome entry site (IRES);
- wherein the first, second, third and optional fourth genes are selected from the group consisting of Oct4, Klf4, Sox2, cMyc, Lin28, and Nanog;
- and wherein the first, second, third and optional fourth genes are not identical,
- wherein if the optional fourth gene is not selected from the group consisting of Oct4, Klf4, Sox2, cMyc, Lin28, and Nanog, a marker gene is included in its place, wherein the marker gene encodes an optically visible protein or an enzyme,
- wherein the nucleic acid sequence is operably linked to a promoter, and
- wherein the sequences encoding the first gene;
- the second gene;
- the third gene;
- the optional fourth gene, first and second ‘self-cleaving’ 2A peptides, and the IRES are transcribed from the promoter as a multi-cistronic RNA.
Dependencies are inferred from the claim text.