Globin gene therapy for treating hemoglobinopathies
The presently disclosed subject matter provides for expression cassettes that allow for expression of a globin gene or a functional portion thereof, vectors comprising thereof, and cells transduced with such expression cassettes and vectors. The presently disclosed subject matter further provides methods for treating a hemoglobinopathy in a subject comprising administering an effective amount of such transduced cells to the subject.
1. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a Dnase I hypersensitive site-2 (HS2) region, a Dnase I hypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4 (HS4) region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO: 50, and wherein the β-globin LCR comprises:
(a) a HS2 region consisting of nucleotides 45-860 of SEQ ID NO: 9, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6;
(b) a HS2 region consisting of the nucleotide sequence set forth in SEQ ID NO: 33, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35;
(c) a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS2 region consisting of nucleotides 45-860 of SEQ ID NO: 9, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6; or
(d) a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS2 region consisting of the nucleotide sequence set forth in SEQ ID NO: 33, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35.
2. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a HS1 region, a HS3 region, and a HS4 region and does not comprise a core sequence of HS2 region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO:50, wherein the β-globin LCR comprises:
(a) a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6; or
(b) a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35.
3. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a Dnase I hypersensitive site-2 (HS2) region, a Dnase I hypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4 (HS4) region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO: 50, wherein the β-globin LCR comprises; a HS2 region consisting of nucleotides 45-860 of SEQ ID NO: 9, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6.
4. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a Dnase I hypersensitive site-2 (HS2) region, a Dnase I hypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4 (HS4) region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO: 50, and wherein the β-globin LCR comprises; a HS2 region consisting of the nucleotide sequence set forth in SEQ ID NO: 33, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35.
5. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a Dnase I hypersensitive site-2 (HS2) region, a Dnase I hypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4 (HS4) region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO: 50, and wherein the β-globin LCR comprises; a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS2 region consisting of nucleotides 45-860 of SEQ ID NO: 9, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6.
6. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a Dnase I hypersensitive site-2 (HS2) region, a Dnase I hypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4 (HS4) region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO: 50, and wherein the β-globin LCR comprises; a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS2 region consisting of the nucleotide sequence set forth in SEQ ID NO: 33, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35.
7. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a Dnase I hypersensitive site-2 (HS2) region, a Dnase I hypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4 (HS4) region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO: 50, and wherein the β-globin LCR consists essentially of; a HS2 region consisting of nucleotides 45-860 of SEQ ID NO: 9, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6.
8. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a Dnase I hypersensitive site-2 (HS2) region, a Dnase I hypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4 (HS4) region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO: 50, and wherein the β-globin LCR consists essentially of; a HS2 region consisting of the nucleotide sequence set forth in SEQ ID NO: 33, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35.
9. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a Dnase I hypersensitive site-2 (HS2) region, a Dnase I hypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4 (HS4) region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO: 50, and wherein the β-globin LCR consists essentially of; a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS2 region consisting of nucleotides 45-860 of SEQ ID NO: 9, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6.
10. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a Dnase I hypersensitive site-2 (HS2) region, a Dnase I hypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4 (HS4) region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO: 50, wherein the β-globin LCR consists essentially of; a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS2 region consisting of the nucleotide sequence set forth in SEQ ID NO: 33, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35.
11. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a HS1 region, a HS3 region, and a HS4 region and does not comprise a core sequence of HS2 region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO:50, and wherein the β-globin LCR comprises; a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6.
12. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a HS1 region, a HS3 region, and a HS4 region and does not comprise a core sequence of HS2 region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO:50, and wherein the β-globin LCR comprises; a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35.
13. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a HS1 region, a HS3 region, and a HS4 region and does not comprise a core sequence of HS2 region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO:50, and wherein the β-globin LCR consists essentially of; a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 5, and a HS4 region consisting of nucleotides 115-868 of SEQ ID NO: 6.
14. An expression cassette comprising a globin gene or a functional portion thereof operably linked to a β-globin locus control region (LCR) that comprises a HS1 region, a HS3 region, and a HS4 region and does not comprise a core sequence of HS2 region, wherein the HS4 region has a length of less than about 800 bp and does not comprise the nucleotide sequence set forth in SEQ ID NO:50, and wherein the β-globin LCR consists essentially of; a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO: 3, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO: 34, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO: 35.