RECOMBINANT VECTORS COMPRISING ARYLSULFATASE A AND THEIR USES IN STEM CELL THERAPY FOR THE TREATMENT OF METACHROMATIC LEUKODYSTROPHY
Provided are recombinant lentiviral vectors comprising an expression cassette comprising a nucleic acid construct expressing an ARSA gene. The vectors are useful in gene therapy for the treatment of Metachromatic Leukodystrophy. Provided are methods of producing the vectors. Provided are multipotent stem cells comprising the vectors. Also provided are methods of culturing the stem cells to maintain their multipotency.
1 . A recombinant lentiviral vector (LV) comprising: an arylsulfatase A (ARSA) gene encoding an arylsulfatase A polypeptide, where said LV is a self-inactivating (SIN) lentiviral vector.
2 . The vector of claim 1 , wherein the vector comprises a recombinant ARSA gene under the control of an ARSA gene 5′ promoter and an ARSA 3′ enhancer.
3 . The vector of claim 1 , wherein the vector comprises an insulator.
4 . The vector of claim 1 , wherein the vector comprises a Rev Responsive Element (RRE).
5 . The vector of claim 1 , wherein the vector comprises a central polypurine tract.
6 . The vector of claim 1 , wherein the vector comprises a post-translational regulatory element.
7 . The vector of claim 6 , wherein the posttranscriptional regulatory element is a modified Woodchuck Post-transcriptional Regulatory Element (WPRE).
8 . The vector of claim 7 , wherein the modified WPRE has a nucleic acid sequence as set forth in SEQ ID NO: 3.
9 . The vector of claim 1 , wherein the ARSA gene is human ARSA gene.
10 . The vector of claim 9 , wherein the ARSA gene has a nucleic acid sequence as set forth in SEQ ID NO: 2.
11 . A host cell transduced with the vector of claim 1 .
12 . The host cell of claim 11 , wherein the cell is a stem cell.
13 . The host cell of claim 12 , wherein said cell is a stem cell derived from bone marrow.
14 . The host cell of claim 11 , wherein the cell is a hematopoietic stem cell.
15 . A method of maintaining the multipotency and increased sensitivity for LV infection of the host cell of claim 11 , comprising incubating the host cells in a media comprising TPO, SCF, FLT3, and IL-3 factors.
16 . The method of claim 15 wherein the media further comprises kinase inhibitors that inhibit mTOR and ROCK activity.
17 . The method of claim 15 wherein the media comprises CP1, glucose, albumin at 2-20% and Herceptin.
18 . The method of claim 17 wherein the albumin is at 5-10%.
19 . The medium of claim 17 , wherein the medium is frozen.
20 . A method of treating Metachromatic leukodystrophy (MLD) in a subject, said method comprising: transducing a stem cell from said subject with a vector of claim 1 ; and transplanting said transduced cell into said subject where said cells express said ARSA gene.
21 . The method of claim 20 wherein the cell is a stem cell derived from bone marrow.
22 . The method of claim 20 wherein the cell is a human hematopoietic stem or progenitor cell.
23 . The method of claim 22 , wherein the method increases the chances of survival of the hematopoietic stem or progenitor cell during gene therapy.
24 . The method of claim 22 , wherein the method prevents apoptosis of the hematopoietic stem or progenitor cell.
25 . A method of regulating expression of a transgene comprising delivering the vector of claim 1 to a hematopoietic stem or progenitor cell.
26 . A pharmaceutical composition comprising the vector of claim 1 for treating Metachromatic leukodystrophy (MLD) in a subject.