MICROGLIA SPECIFIC PROMOTERS AND METHODS OF USE THEREFORE
20220348962 · 2022-11-03
Assignee
- Children's Medical Center Corporation (Boston, MA)
- Dana-Farber Cancer Institute, Inc. (Boston, MA, US)
Inventors
- Alessandra Biffi (Boston, MA, US)
- Valentina POLETTI (Boston, MA, US)
- Marco PEVIANI (Boston, MA, US)
- Francisco J. MOLINA-ESTEVEZ (Boston, MA, US)
Cpc classification
C12N2740/15043
CHEMISTRY; METALLURGY
C12N2740/16043
CHEMISTRY; METALLURGY
C12N2830/008
CHEMISTRY; METALLURGY
A61K48/00
HUMAN NECESSITIES
C12N15/63
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure features methods and compositions for expressing transgenes in microglia. The disclosed compositions comprise isolated fragments of human and murine translocator protein (TSPO) promoters and expression cassettes comprising the same. The methods involve using these promoters and/or expression cassettes to express transgenes in a cell.
Claims
1. An isolated translocator protein (TSPO) promoter fragment sufficient to direct expression of a downstream polynucleotide in a microglial cell.
2. The isolated TSPO promoter fragment of claim 1, wherein the TSPO promoter fragment is responsive to inflammation.
3. The isolated TSPO promoter fragment of claim 1, wherein the fragment is selected from the group consisting of about 300, 1200, 1600 nucleotides of a TSPO promoter.
4. The isolated TSPO promoter fragment of claim 1, wherein the fragment comprises or consists of at least about 300 nucleotides.
5. The isolated TSPO promoter fragment of claim 1, wherein the promoter fragment has at least about 85% nucleotide sequence identity to the sequence of MPP01, MPP02, MPP03, or MPP04.
6. The isolated TSPO promoter fragment of claim 1, wherein the full-length promoter comprises nucleotides 83,518,714 to 83,521,414 of murine chromosome 15 or a corresponding portion of a human chromosome.
7-8. (canceled)
9. The isolated TSPO promoter fragment of claim 1, wherein the promoter fragment comprises or consists essentially of a polynucleotide selected from the group consisting of: TABLE-US-00010 >MPP01 (hTSPO_prximal5′prom)(“Pl”) Tgcatcaccgcgttgcggcctcatcagtcccacgactttgtgcccattttactcatgaggagatggaggcccagagagccagtc agaaagtggctgggccaggactaagagtgcagcgcgctgcctccgtgccctgcgtcaacagctcaaggaactggggtgctcc ggaaatggggccaaggctgctgggcagcaggacgctcagggccttggcctcaggagagcaaattccccactcggagatcgg tcttgttgctgcattttattcatgggaaatctgaggctagaagagacgacaaacgacacgccgttggacacacggcaacgttttag atgttgggtctggccgggcggccgtcaccggtcaccatggggaggaggaggagccgagagacttgctcgcggccgggggg aggcagaagcgcgtcccgcgggagaggtggctttgaggagtgagctcccggtcccgcggggacgcgagtgggcccagtgc ccgggctgccaggcggggcggggcggggccgggcgactgagaggggcggggcctggcggctgggaggggCGGGG CGGATGCGGGGACAGCGGCCTGGCTAACTCCTGCACGGCAGTGCCCTTCCCG GAGCGTGCCCTCGCCG >MPP02(hTSPO_intronic5′prom)(“P2”) CTGcacagtgaggacgggacgcggagggggcagcgggaacacgccgcccgcatggctgcgacagttggcagcgccgc gggacagagggaaactgaggccggagccgcagactggacacccgagggggcgacccggggcagcacttggggctcggc tacgcgcacagggggcggcgggcagcagagtctgggcctccgcggccggggttccaccgccggccgcctccggctcgcg caacgggagggaaaacttggacaaccctgccacgcccagcccttggccgcgtggcttctcctgctcgaagcgcggtcccagg agtggccgacgctccctctcctgcccattccgcggatgggcaatcccaggcggaactcccttgagggtctcagaatatctggga gacctcgggctcttgatctccgagacaccccgtttcgtagtggagaacagtccagatcggggaagtttattttgcccaaagccgc atagaggccccctggccctcgattccctctgcggggctcagcagcgttgcagcctagacgggtcttactgtgagccgagcagc ctctgggaccacagaccttcccctaccccaacgttagaagccggagcccagcaaggagaagcgcgcacctcctgctgtgaac gcgcacgacgccagggcagctgccagaggccatggcctggcgtgggcctggagcccctctggccagcctgcacggggcca gggctacgggataccagcagcgtgccctgggctggatggcaggagagacaggacttgaggctgtcccagaatgggctcagg cagggcgaggatatcaggggaggtggtgtacaggaagcagccgcccagcttgcctggcacacagcaagccctgcccatgaa ggcctactgccagaacagtgggcgaggcccggcgtctctgtggagtcggtggggcccgggacagggcagcctgaggcagg tttccactggcggtgaaaggggccgtgtggcaaggacaggagagccagcctcagcccagcaggggaaggcggcccctgag tctccacctggctgctggcagccccactcggagcatcggcgaaactgaggcttgccaaagaagcctttgtccagagtcacgca gctggcgcggtggagccagggccagaacccgtgcaggctgatcccagcctgccttctccactgtgccccg >MPP03(hTSPO_upstream_plus_intronic_prom)(“P1 + P2”) GAGtgcatcaccgcgttgcggcctcatcagtcccacgactttgtgcccattttactcatgaggagatggaggcccagagagcc agtcagaaagtggctgggccaggactaagagtgcagcgcgctgcctccgtgccctgcgtcaacagctcaaggaactggggtg ctccggaaatggggccaaggctgctgggcagcaggacgctcagggccttggcctcaggagagcaaattccccactcggagat cggtcttgttgctgcattttattcatgggaaatctgaggctagaagagacgacaaacgacacgccgttggacacacggcaacgttt tagatgttgggtctggccgggcggccgtcaccggtcaccatggggaggaggaggagccgagagacttgctcgcggccggg gggaggcagaagcgcgtcccgcgggagaggtggctttgaggagtgagctcccggtcccgcggggacgcgagtgggccca gtgcccgggctgccaggcggggcggggcggggccgggcgactgagaggggcggggcctggcggctgggaggggCG GGGCGGATGCGGGGACAGCGGCCTGGCTAACTCCTGCACGGCAGTGCCCTTC CCGGAGCGTGCCCTCGCCGCTGcacagtgaggacgggacgcggagggggcagcgggaacacgccgcc cgcatggctgcgacagttggcagcgccgcgggacagagggaaactgaggccggagccgcagactggacacccgaggggg cgacccggggcagcacttggggctcggctacgcgcacagggggcggcgggcagcagagtctgggcctccgcggccgggg ttccaccgccggccgcctccggctcgcgcaacgggagggaaaacttggacaaccctgccacgcccagcccttggccgcgtg gcttctcctgctcgaagcgcggtcccaggagtggccgacgctccctctcctgcccattccgcggatgggcaatcccaggcgga actcccttgagggtctcagaatatctgggagacctcgggctcttgatctccgagacaccccgtttcgtagtggagaacagtccag atcggggaagtttattttgcccaaagccgcatagaggccccctggccctcgattccctctgcggggctcagcagcgttgcagcct agacgggtcttactgtgagccgagcagcctctgggaccacagaccttcccctaccccaacgttagaagccggagcccagcaa ggagaagcgcgcacctcctgctgtgaacgcgcacgacgccagggcagctgccagaggccatggcctggcgtgggcctgga gcccctctggccagcctgcacggggccagggctacgggataccagcagcgtgccctgggctggatggcaggagagacagg acttgaggctgtcccagaatgggctcaggcagggcgaggatatcaggggaggtggtgtacaggaagcagccgcccagcttg cctggcacacagcaagccctgcccatgaaggcctactgccagaacagtgggcgaggcccggcgtctctgtggagtcggtgg ggcccgggacagggcagcctgaggcaggtttccactggcggtgaaaggggccgtgtggcaaggacaggagagccagcctc agcccagcaggggaaggcggcccctgagtctccacctggctgctggcagccccactcggagcatcggcgaaactgaggctt gccaaagaagcctttgtccagagtcacgcagctggcgcggtggagccagggccagaacccgtgcaggctgatcccagcctg ccttctccactgtgccccg >MPP04(hTSPO_intronic_plus_upstream_prom)(“P2 + Pl”) CTGcacagtgaggacgggacgcggagggggcagcgggaacacgccgcccgcatggctgcgacagttggcagcgccgc gggacagagggaaactgaggccggagccgcagactggacacccgagggggcgacccggggcagcacttggggctcggc tacgcgcacagggggcggcgggcagcagagtctgggcctccgcggccggggttccaccgccggccgcctccggctcgcg caacgggagggaaaacttggacaaccctgccacgcccagcccttggccgcgtggcttctcctgctcgaagcgcggtcccagg agtggccgacgctccctctcctgcccattccgcggatgggcaatcccaggcggaactcccttgagggtctcagaatatctggga gacctcgggctcttgatctccgagacaccccgtttcgtagtggagaacagtccagatcggggaagtttattttgcccaaagccgc atagaggccccctggccctcgattccctctgcggggctcagcagcgttgcagcctagacgggtcttactgtgagccgagcagc ctctgggaccacagaccttcccctaccccaacgttagaagccggagcccagcaaggagaagcgcgcacctcctgctgtgaac gcgcacgacgccagggcagctgccagaggccatggcctggcgtgggcctggagcccctctggccagcctgcacggggcca gggctacgggataccagcagcgtgccctgggctggatggcaggagagacaggacttgaggctgtcccagaatgggctcagg cagggcgaggatatcaggggaggtggtgtacaggaagcagccgcccagcttgcctggcacacagcaagccctgcccatgaa ggcctactgccagaacagtgggcgaggcccggcgtctctgtggagtcggtggggcccgggacagggcagcctgaggcagg tttccactggcggtgaaaggggccgtgtggcaaggacaggagagccagcctcagcccagcaggggaaggcggcccctgag tctccacctggctgctggcagccccactcggagcatcggcgaaactgaggcttgccaaagaagcctttgtccagagtcacgca gctggcgcggtggagccagggccagaacccgtgcaggctgatcccagcctgccttctccactgtgccccgtgcatcaccgcgt tgcggcctcatcagtcccacgactttgtgcccattttactcatgaggagatggaggcccagagagccagtcagaaagtggctgg gccaggactaagagtgcagcgcgctgcctccgtgccctgcgtcaacagctcaaggaactggggtgctccggaaatggggcca aggctgctgggcagcaggacgctcagggccttggcctcaggagagcaaattccccactcggagatcggtcttgttgctgcatttt attcatgggaaatctgaggctagaagagacgacaaacgacacgccgttggacacacggcaacgttttagatgttgggtctggcc gggcggccgtcaccggtcaccatggggaggaggaggagccgagagacttgctcgcggccggggggaggcagaagcgcg tcccgcgggagaggtggctttgaggagtgagctcccggtcccgcggggacgcgagtgggcccagtgcccgggctgccagg cggggcggggcggggccgggcgactgagaggggcggggcctggcggctgggaggggCGGGGCGGATGCG GGGACAGCGGCCTGGCTAACTCCTGCACGGCAGTGCCCTTCCCGGAGCGTGC CCTCGCCGGGATCC
10-11. (canceled)
12. An expression vector comprising the promoter fragment of claim 1.
13-17. (canceled)
18. The expression vector of claim 17, wherein the vector further comprises an enhancer that comprises or consists essentially of E1, E1.1, E1.2, or E2.
19. An expression vector comprising a regulatory sequence driving expression of a downstream gene, the regulatory sequence selected from the group consisting of P1, from 5′ to 3′ P1 and P2, from 5′ to 3′ P2 and P1, from 5′ to 3′ El and P1, and 5′ to 3′ E2 and P1.
20-23. (canceled)
24. An expression vector comprising a regulatory element comprising a polynucleotide sequence having at least 85% identity to any of the following sequences: TABLE-US-00011 >MPP01(hTSPO_prximal5′prom)(“Pl”) Tgcatcaccgcgttgcggcctcatcagtcccacgactttgtgcccattttactcatgaggagatggaggcccagagagccagtc agaaagtggctgggccaggactaagagtgcagcgcgctgcctccgtgccctgcgtcaacagctcaaggaactggggtgctcc ggaaatggggccaaggctgctgggcagcaggacgctcagggccttggcctcaggagagcaaattccccactcggagatcgg tcttgttgctgcattttattcatgggaaatctgaggctagaagagacgacaaacgacacgccgttggacacacggcaacgttttag atgttgggtctggccgggcggccgtcaccggtcaccatggggaggaggaggagccgagagacttgctcgcggccgggggg aggcagaagcgcgtcccgcgggagaggtggctttgaggagtgagctcccggtcccgcggggacgcgagtgggcccagtgc ccgggctgccaggcggggcggggcggggccgggcgactgagaggggcggggcctggcggctgggaggggCGGGG CGGATGCGGGGACAGCGGCCTGGCTAACTCCTGCACGGCAGTGCCCTTCCCG GAGCGTGCCCTCGCCG. >MPP02(hTSPO_intronic5'prom)(“P2”) CTGcacagtgaggacgggacgcggagggggcagcgggaacacgccgcccgcatggctgcgacagttggcagcgccgc gggacagagggaaactgaggccggagccgcagactggacacccgagggggcgacccggggcagcacttggggctcggc tacgcgcacagggggcggcgggcagcagagtctgggcctccgcggccggggttccaccgccggccgcctccggctcgcg caacgggagggaaaacttggacaaccctgccacgcccagcccttggccgcgtggcttctcctgctcgaagcgcggtcccagg agtggccgacgctccctctcctgcccattccgcggatgggcaatcccaggcggaactcccttgagggtctcagaatatctggga gacctcgggctcttgatctccgagacaccccgtttcgtagtggagaacagtccagatcggggaagtttattttgcccaaagccgc atagaggccccctggccctcgattccctctgcggggctcagcagcgttgcagcctagacgggtcttactgtgagccgagcagc ctctgggaccacagaccttcccctaccccaacgttagaagccggagcccagcaaggagaagcgcgcacctcctgctgtgaac gcgcacgacgccagggcagctgccagaggccatggcctggcgtgggcctggagcccctctggccagcctgcacggggcca gggctacgggataccagcagcgtgccctgggctggatggcaggagagacaggacttgaggctgtcccagaatgggctcagg cagggcgaggatatcaggggaggtggtgtacaggaagcagccgcccagcttgcctggcacacagcaagccctgcccatgaa ggcctactgccagaacagtgggcgaggcccggcgtctctgtggagtcggtggggcccgggacagggcagcctgaggcagg tttccactggcggtgaaaggggccgtgtggcaaggacaggagagccagcctcagcccagcaggggaaggcggcccctgag tctccacctggctgctggcagccccactcggagcatcggcgaaactgaggcttgccaaagaagcctttgtccagagtcacgca gctggcgcggtggagccagggccagaacccgtgcaggctgatcccagcctgccttctccactgtgccccg >E1 agactctgtctcaaaaaaaaaaaaaaaaaaaaaaaaagagagagagaaaaaggaagttagaaaaacagccctagaggcccta cattctgagtaataggagttccagaaaggaagtgattgctgcacaacataaatttgaaaagaaagagaagtgagaaaatagagg gaaggaaatcaaagaaataatccaacttctgaaaagtaaagaatgagcttccagcgggaaagtgcctgttgagtgcaggcaca gtggaggaaatgaagctgggtgtgttgccaggagttggaaacttgtctgagcaacatagcgcgaccctgtgtctacaaaaaaat aaaaacaaaacaaaaaacaaccaaagacttccgaaacagaatggctttagcctgctcaaccgcacactggcacctggccaaca gcatctcttcatgattctgaaggacaacgatctgcagctcagccaagcatcagccatctatggcctaggatgcaagaattcagca atgttaccttcAgactctgtctcaaaaaaaaaaaaaaaaaaaaaaaaagagagagagaaaaaggaagttagaaaaacagccct agaggccctacattctgagtaataggagttccagaaaggaagtgattgctgcacaacataaatttgaaaagaaagagaagtgag aaaatagagggaaggaaatcaaagaaataatccaacttctgaaaagtaaagaatgagcttccagcgggaaagtgcctgttgagt gcaggcacagtggaggaaatgaagctgggtgtgttgccaggagttggaaacttgtctgagcaacatagcgcgaccctgtgtcta caaaaaaataaaaacaaaacaaaaaacaaccaaagacttccgaaacagaatggctttagcctgctcaaccgcacactggcacc tggccaacagcatctcttcatgattctgaaggacaacgatctgcagctcagccaagcatcagccatctatggcctaggatgcaag aattcagcaatgttaccttctgcatcaccgcgttgcggcctcatcagtcccacgactttgtgcccattttactcatgaggagatggag gcccagagagccagtcagaaagtggctgggccaggactaagagtgcagcgcgctgcctccgtgccctgcgtcaacagctca aggaactggggtgctccggaaatggggccaaggctgctgggcagcaggacgctcagggccttggcctcaggagagcaaatt ccccactcggagatcggtcttgttgctgcattttattcatgggaaatctgaggctagaagagacgacaaacgacacgccgttgga cacacggcaacgttttagatgttgggtctggccgggcggccgtcaccggtcaccatggggaggaggaggagccgagagactt gctcgcggccggggggaggcagaagcgcgtcccgcgggagaggtggctttgaggagtgagctcccggtcccgcggggac gcgagtgggcccagtgcccgggctgccaggcggggcggggcggggccgggcgactgagaggggcggggcctggcggc tgggaggggCGGGGCGGATGCGGGGACAGCGGCCTGGCTAACTCCTGCACGGCA GTGCCCTTCCCGGAGCGTGCCCTCGCCG >E2 Tgcatcaccgcgttgcggcctcatcagtcccacgactttgtgcccattttactcatgaggagatggaggcccagagagccagtc agaaagtggctgggccaggactaagagtgcagcgcgctgcctccgtgccctgcgtcaacagctcaaggaactggggtgctcc ggaaatggggccaaggctgctgggcagcaggacgctcagggccttggcctcaggagagcaaattccccactcggagatcgg tcttgttgctgcattttattcatgggaaatctgaggctagaagagacgacaaacgacacgccgttggacacacggcaacgttttag atgttgggtctggccgggcggccgtcaccggtcaccatggggaggaggaggagccgagagacttgctcgcggccgggggg aggcagaagcgcgtcccgcgggagaggtggctttgaggagtgagctcccggtcccgcggggacgcgagtgggcccagtgc ccgggctgccaggcggggcggggcggggccgggcgactgagaggggcggggcctggcggctgggaggggCGGGG CGGATGCGGGGACAGCGGCCTGGCTAACTCCTGCACGGCAGTGCCCTTCCCG GAGCGTGCCCTCGCCG. >MPP05A(hTSPO upstream enhancer)(“E1.1”) agactctgtctcaaaaaaaaaaaaaaaaaaaaaaaaagagagagagaaaaaggaagttagaaaaacagccctagaggcccta cattctgagtaataggagttccagaaaggaagtgattgctgcacaacataaatttgaaaagaaagagaagtgagaaaatagagg gaaggaaatcaaagaaataatccaacttctgaaaagtaaagaatgagcttccagcgggaaagtgcctgttgagtgcaggcaca gtggaggaaatgaagctgggtgtgttgccaggagttggaaacttgtctgagcaacatagcgcgaccctgtgtctacaaaaaaat aaaaacaaaacaaaaaacaaccaaagacttccgaaacagaatggctttagcctgctcaaccgcacactggcacctggccaaca gcatctcttcatgattctgaaggacaacgatctgcagctcagccaagcatcagccatctatggcctaggatgcaagaattcagca atgttaccttc >MPP05B(hTSPO_upstream_enhancer)(“E1.2”) Agactctgtctcaaaaaaaaaaaaaaaaaaaaaaaaagagagagagaaaaaggaagttagaaaaacagccctagaggccct acattctgagtaataggagttccagaaaggaagtgattgctgcacaacataaatttgaaaagaaagagaagtgagaaaatagag ggaaggaaatcaaagaaataatccaacttctgaaaagtaaagaatgagcttccagcgggaaagtgcctgttgagtgcaggcac agtggaggaaatgaagctgggtgtgttgccaggagttggaaacttgtctgagcaacatagcgcgaccctgtgtctacaaaaaaa taaaaacaaaacaaaaaacaaccaaagacttccgaaacagaatggctttagcctgctcaaccgcacactggcacctggccaac agcatctcttcatgattctgaaggacaacgatctgcagctcagccaagcatcagccatctatggcctaggatgcaagaattcagc aatgttaccttctgcatcaccgcgttgcggcctcatcagtcccacgactttgtgcccattttactcatgaggagatggaggcccaga gagccagtcagaaagtggctgggccaggactaagagtgcagcgcgctgcctccgtgccctgcgtcaacagctcaaggaactg gggtgctccggaaatggggccaaggctgctgggcagcaggacgctcagggccttggcctcaggagagcaaattccccactc ggagatcggtcttgttgctgcattttattcatgggaaatctgaggctagaagagacgacaaacgacacgccgttggacacacggc aacgttttagatgttgggtctggccgggcggccgtcaccggtcaccatggggaggaggaggagccgagagacttgctcgcgg ccggggggaggcagaagcgcgtcccgcgggagaggtggctttgaggagtgagctcccggtcccgcggggacgcgagtgg gcccagtgcccgggctgccaggcggggcggggcggggccgggcgactgagaggggcggggcctggcggctgggaggg gCGGGGCGGATGCGGGGACAGCGGCCTGGCTAACTCCTGCACGGCAGTGCCC TTCCCGGAGCGTGCCCTCGCCG
25. A mammalian cell comprising the expression vector of claim 12.
26-27. (canceled)
28. A method for expressing a therapeutic polypeptide in a cell, the method comprising contacting the cell with an expression vector comprising the promoter fragment of claim 1.
29. (canceled)
30. A method of treating a subject having a neurological disease, the method comprising contacting a cell of the subject with an expression vector comprising the promoter fragment of claim 1 or administering to the subject a cell comprising an expression vector having the promoter fragment of claim 1.
31. (canceled)
32. The method of claim 30, wherein the neurological disease is Alzheimer's disease, amyotrophic lateral sclerosis, metachromatic leuokodystrophy, adrenoleukodystophy, a lysosomal storage disorder, or Parkinson's disease.
33-36. (canceled)
37. The method of claim 30, wherein the promoter fragment comprises or consists essentially of P1 or P1+P2.
38. (canceled)
39. The method of claim 30, wherein the vector further comprises an enhancer comprising or consisting essentially of E1, E2m .
40-41. (canceled)
42. The method of claim 30, wherein the cell is a hematopoietic stem cell or progenitor thereof or a microglial cell.
43. The method of claim 30, wherein the subject undergoes ablative conditioning prior to the method.
44. (canceled)
48. The method of claim 30, wherein the cell is a cell in vitro or in vivo.
49. A pharmaceutical composition comprising the expression vector of claim 12 or a cell comprising said vector.
50. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0101] Efficient expression of therapeutic transgenes in cells residing in the central nervous system can potentially ameliorate, or even cure, neurodegenerative disease. Referring to
[0102] Not intending to be bound by theory, neuroinflammation is largely driven by microglial cell-activation and plays a major role in the pathogenesis and progression of Lysosomal Storage Disorders (LSD), among other neurodegenerative diseases. The beneficial effect of ex vivo gene therapy (GT) on lysosomal storage disorder (LSD) patients relies, at least in part, on the normalization of microglial cell homeostasis and reduction of neuroinflammation after the treatment. In some embodiments of a gene therapy (GT) for lysosomal storage disorders (LSDs), enzyme-competent microglial cells derived from genetically-modified hematopoietic stem cells (HSCs) engraft into a patient's Central Nervous System (CNS) where they release therapeutic factors to be available for all the resident CNS cells. Microglia-mediated delivery of immunomodulatory or other anti-oxidant factors could be used to mitigate neuroinflammation and neurodegeneration.
[0103] A suitable lentiviral vector for a gene therapy (GT) strategy to treat neurodegenerative diseases in various embodiments can drive regulated expression of the therapeutic transgene in hematopoietic stem progenitor cell-derived (HSPC-derived) microglial cells: upregulated in the presence of neuroinflammation and downregulated under homeostatic central nervous system (CNS) conditions. Such a transcriptional pattern is typical of the human translocator protein (TSPO) gene. Its overexpression is a hallmark of in vivo microglial activation in neurodegenerative disorders, suggesting TSPO promoter may be characterized by a minimal basal activity highly-inducible upon cell activation. The examples provided herein present an analysis used to identify regulatory elements that control this transcriptional pattern of a putative regulatory region of the human TSPO gene by integrating publicly-available transcriptional and epigenetic data produced by the Broad Institute-Encode Project in human hematopoietic cell lines (K562 cells). By a deep in silico analysis of the genomic region upstream and downstream to the human translocator protein (TSPO) transcriptional start site, regions were identified that were epigenetically marked as active promoters (H3K4me3.sup.+ and H3K27ac.sup.+) or active enhancers (H3K4me1.sup.+ and H3K27ac.sup.+) bound by hematopoietic-specific transcription factors as detected by Chip-seq genomic maps. Epigenetically-identified promoter regions were defined as actual promoters if active transcription was confirmed by RNA-seq data.
[0104] In the examples provided herein, four different putative regulatory elements were identified by this analysis (P1, P2, E1, and E2), which were subsequently modified for use in lentiviral vectors: small size, and removal of canonical and cryptic poly-adenylation and splicing signals. After these ad hoc modifications, putative regulatory elements were combined in different activation-responsive regulatory cassettes, which were cloned in five corresponding lentiviral vectors (LV MPP01-03-04-07-08) to control the expression of a Green Fluorescent Protein (GFP) reporter gene. These lentiviral vectors (LVs) were produced at a high titer and infectivity, and are currently under preclinical investigation in human microglial cells (HMC3 cell line) to test their transcriptional level under basal and cell-activation conditions. In various embodiments, the best vector(s) may be suitable for treatment of human neurodenerative diseases on account of their ability to safely and specifically induce a therapeutic level of transgene expression in pathological central nervous system (CNS) conditions.
[0105] Translocator protein (TPSO) is an outer mitochondrial membrane protein expressed in microglia cells and other phagocytic immune cells of the central nervous system. Expression of the TSPO gene is upregulated in microglia activated in response to pathological events and by lipopolysaccharide (LPS), a glycoprotein on the cell walls of Gram-negative bacteria (Visigalli et al. Neurobiol Dis 34, 51-62 (2009); Turner et al. Neurobiol Dis 15, 601-609 (2004), the contents of which are incorporated by reference in their entirety). The full-length TSPO promoter has been used to express transgenes in microglia, as described in PCT/US2017/056774, the contents of which are incorporated herein by reference in their entirety.
[0106] The full-length TSPO promoter is about 2700 nucleotides, which represents a significant percentage of the packaging capacity of most viral vectors. To compensate for the limited packaging capacity of viral vectors, some aspects of the present disclosure provide nucleic acid molecules comprising a truncated TSPO promoter operably linked to a coding sequence of a polynucleotide or polypeptide. The nucleic acid molecules are plasmids in some embodiments. Plasmids, such as cloning vectors, can be used to manufacture many copies of a transgene cassette that comprises a truncated TSPO promoter operably linked to a coding sequence. The plasmid may be an expression vector that can be used in an in vitro or in vivo environment to express the encoded transgene. In some embodiments, the nucleic acid molecules comprising a truncated TSPO promoter operably linked to a coding sequence of a polynucleotide or polypeptide is an isolated nucleic acid molecule.
[0107] In some embodiments, the truncated TSPO promoter comprises a nucleic acid having 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, or even 2400 fewer nucleotide than the wildtype TSPO promoter. For example, in some embodiments, the nucleic acid sequence of a truncated TSPO promoter consists of SEQ ID NO. 3 (T1600), SEQ ID NO: 5 (T1200), or SEQ ID NO: T300. In some embodiments, the truncated TSPO promoter is a non-naturally occurring nucleic acid sequence. In some embodiments, the non-naturally occurring nucleic acid sequence of the TSPO promoter is derived from the nucleic acid sequence of the human TSPO promoter. .
[0108] In some embodiments of the present disclosure, cultured cells are transfected with an expression vector comprising a transgene, wherein the transgene includes a truncated TSPO promoter operably linked to a coding sequence to express the encoded polynucleotide or polypeptide. To efficiently induce the cells to express the transgene's encoded polynucleotide or polypeptide, the cells are contacted with an activating agent, including but not limited to LPS. In some embodiments, the cells are stably transfected with a plasmid comprising the transgene as well as additional nucleic acid sequences that promote integration into a recipient cell's genome of a coding sequence of a transgene operably linked to a truncated TSPO promoter. In some embodiments, microglia cells, or precursors thereof, are transduced. In some embodiments, hematopoietic stem progenitor cells (HSPCs), or progeny thereof, are transduced. Methods of transfecting cells in a culture are well-known in the art.
[0109] In some embodiments of the present disclosure, cells may be transduced with a viral vector comprising a transgene operably linked to a truncated TSPO promoter is packaged within the vector. To transduce cells, viral vectors are assembled, and the nucleic acid molecule comprising a truncated TSPO promoter operably linked to a coding sequence are packaged within the vector. Because of the limited packaging capacity of viral vectors, the presently disclosed truncated TSPO promoter system has distinct advantages over previous systems using full-length promoters. By employing a truncated TSPO promoter, a larger coding sequence or additional regulatory sequences can be included in the transgene.
[0110] Viral vectors suitable for transducing microglia, or precursors thereof, or HSPCs, or progeny thereof, include but are not limited to lentiviral vectors, gamma-retroviral vectors, adenoviral vectors, and adeno-associated viral vectors. These vector systems are well-known in the art. In some embodiments, avian viral vectors are used to transduce microglia, or precursors thereof, or HSPCs, or progeny thereof. Avian viral vector systems include those described in U.S. Pat. No. 8,642,570, DE102009021592, PCT/EP2010/056757, and EP2430167, the contents of which are incorporated herein by reference in their entirety.
[0111] In some embodiments of the present disclosure, the transgene encodes a reporter polypeptide comprising detectable label. Examples of a reporter peptide include, but are not limited to, green fluorescent protein (GFP) and blue fluorescent protein (BFP). In some embodiments, expression of an expressed transgene is detected by contacting the cell with a detectably labeled reagent that specifically binds the expressed polynucleotide or polypeptide. In some embodiments, the reagent that specifically binds an expressed polypeptide is an antibody. Methods for producing antibodies and detectably labeled antibodies are known in the art. In some embodiments, the reagent that specifically binds an expressed nucleic acid is a detectably labeled polynucleotide comprising a nucleic acid sequence complementary to the expressed nucleic acid molecule. Detectable labels can be used to identify modified cells expressing the coding sequence of the transgene.
[0112] The present disclosure also includes vectors (e.g., recombinant plasmids) that include nucleic acid molecules (e.g., transgenes) as described herein. The term “recombinant vector” includes a vector (e.g., plasmid, phage, phasmid, virus, cosmid, fosmid, or other purified nucleic acid vector) that has been altered, modified or engineered such that it contains greater, fewer or different nucleic acid sequences than those included in the native or natural nucleic acid molecule from which the recombinant vector was derived. For example, a recombinant vector may include the nucleotide sequence comprising the TSPO promoter operably linked to a coding sequence. The vector may include additional regulatory sequences such as terminator sequences, long terminal repeats, untranslated regions, and the like. Recombinant expression vectors allow for expression of the genes or nucleic acids included in them.
[0113] Methods of introducing exogenous nucleic acid molecules into a cell (e.g., cells comprising a transgene having a truncated TSPO promoter) are known in the art. For example, eukaryotic cells can take up nucleic acid molecules from the environment via transfection (e.g., calcium phosphate-mediated transfection). “Stable transfection” refers to integration of the transfected nucleic acid into the recipient cell's genome, thereby allowing transmission of the transfected nucleic acid to the recipient cell's progeny. In some embodiments of the present disclosure, a nucleic acid molecule comprising a transgene as described herein comprises a nucleotide sequence that facilitates integration of the nucleic acid molecule into genome of a cell. Cells which have been stably transfected or transduced by the introduced DNA comprising a truncated TSPO promoter can be selected, for example, by including coding sequences in the vector for one or more markers that allow for selection of host cells which contain the expression vector. The markers can be a selective marker or a screenable marker.
[0114] In some embodiments, are modified via transduction. Transduction of most cell types can be accomplished with retroviral, lentiviral, adenoviral, adeno-associated, and avian virus systems, and such systems are well-known in the art. While retroviral systems are generally not compatible with neuronal cell transduction, lentiviruses are well-suited for transducing stem cells as well as neuronal cells. Thus, in some embodiments of the present disclosure, the viral vector system is a lentiviral system.
[0115] In some embodiments, the viral vector system is an avian virus system, for example, the avian viral vector system described in U.S. Pat. No. 8,642,570, DE102009021592, PCT/EP2010/056757, and EP2430167, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the viral vectors are assembled or packaged in a packaging cell prior to contacting the intended recipient cell. In some embodiments, the vector system is a self-inactivating system, wherein the viral vector is assembled in a packaging cell, but after contacting the recipient cell, the viral vector is not able to be produced in the recipient cell. The components of a viral vector are encoded on plasmids, and because efficiencies of transduction decrease with large plasmid size, multiple plasmids that have different viral sequences necessary for packaging may be necessary. For example, in a lentiviral vector system, a first plasmid may comprise a nucleotide sequence encoding a Group antigens (gag) and/or a reverse transcriptase (pol) gene, while a second plasmid encodes regulator of expression of virion proteins (rev) and/or envelope (env) genes. The exogenous nucleic acid molecule comprising a transgene can be packaged into the vector and delivered into a recipient cell where the transgene is integrated into the recipient cell's genome. Additionally, the transgene may be packaged using a split-packaging system as described in U.S. Pat. No. 8,642,570, DE102009021592, PCT/EP2010/056757, and EP2430167.
[0116] In some embodiments, after the introduction of one or more vector(s), host cells are cultured prior to administration to a subject. Expression of recombinant proteins encoded in the vectors can be detected by immunoassays including Western blot analysis, immunoblot, and immunofluorescence.
Hematopoietic Cell Transplantation
[0117] In some embodiments, a cell, such as HSPCs and/or their progeny, that comprise a transgene having a truncated TSPO promoter can serve as vehicles for therapeutic molecule delivery across the blood brain barrier by contributing to the turnover of myeloid cell populations in the brain. Methods for hematopoietic cell transplantation (HCT) and hematopoietic stem cell (HSC)-based gene therapy are known in the art and have been used to treat patients affected by non-hematological and non-oncological diseases affecting the nervous system, such as peroxisomal disorders and lysosomal storage diseases (LSDs) (Cartier et al. Science 326, 818-823 (2009); Biffi et al. Science 341, 1233158 (2013); Sessa et al. Lancet 388, 476-487 (2016)) and neurological diseases (Simard et al. Neuron 49, 489-502 (2006)). Indeed, HSPCs and/or their progeny can contribute to the turnover of myeloid cell populations in the brain (Ajami et al. Nat Neurosci 10, 1538-1543 (2007); Ajami et al. Nat Neurosci 14, 1142-1149 (2011); Biffi et al. J. Clin. Invest. 116, 3070-3082 (2006); Mildner et al. Nat Neurosci 10, 1544-1553 (2007); Capotondo et al. Proc Natl Acad Sci USA. 109, 15018-15023 (2012). Microglia's role in the progression and outcomes of these disorders has been described (Jeyakumar et al. Brain 126, 974-987 (2003); Wada et al. Proc Natl Acad Sci USA 97, 10954-10959 (2000); Ohmi et al. Proc. Natl. Acad. Sci. USA 100, 1902-1907 (2003); Eichler et al. Ann Neurol 63, 729-742 (2008), the contents of each are incorporated herein in their entirety).
[0118] Transplant efficiency can be improved with an ablative preconditioning regimen to destroy endogenous microglia, such as that described in International Application No. PCT/US2017/056774, the contents of which are incorporated herein by reference in their entirety. In some embodiments of the present disclosure, the ablative conditioning regimen comprises administering an alkylating agent to a subject prior to transplantation. In some embodiments, the alkylating agent is busulfan. Busulfan is capable of ablating functionally-defined brain-resident microglia precursors (Capotondo et al. Proc Natl Acad Sci USA. 109, 15018-15023 (2012); Wilkinson et al. Mol Ther 21, 868-876 (2013)). In addition to alkylation agents, such as busulfan, CSF-1R inhibitors (e.g., PLX3397 and PLX5622), and liposomal clodronate may be used (Han et al. Molecular Brain, 10:25, 2017), optionally they may be used in combination with the nanoparticles described, for example, in WO2019191650, which is incorporated herein.
[0119] In general, the term nanoparticle refers to any particle having a diameter of less than 1000 nm. In certain preferred embodiments, nanoparticles of the invention have a greatest dimension (e.g., diameter) of 500 nm or less. In other preferred embodiments, nanoparticles of the invention have a greatest dimension ranging between 25 nm and 200 nm. In other preferred embodiments, nanoparticles of the invention have a greatest dimension of 100 nm or less. In other preferred embodiments, nanoparticles of the invention have a greatest dimension ranging between 35 nm and 60 nm. Nanoparticles encompassed in the present invention may be provided in different forms, e.g., as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid-based solids, polymers), suspensions of nanoparticles, or combinations thereof. Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., core-shell nanoparticles). Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically sub 10 nm) that quantization of electronic energy levels occurs.
[0120] Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present invention. Semi-solid and soft nanoparticles have been manufactured, and are within the scope of the present invention. A prototype nanoparticle of semi-solid nature is the liposome. Various types of liposome nanoparticles are currently used clinically as delivery systems for anticancer drugs and vaccines. Nanoparticles with one half hydrophilic and the other half hydrophobic are termed Janus particles and are particularly effective for stabilizing emulsions. They can self-assemble at water/oil interfaces and act as solid surfactants. In one embodiment, nanoparticles based on self-assembling bioadhesive polymers are contemplated, which may be applied to oral delivery of agents, intravenous delivery of agents and nasal delivery of agents, all to the brain. Other embodiments, such as oral absorption and ocular deliver of hydrophobic drugs are also contemplated. The molecular envelope technology involves an engineered polymer envelope which is protected and delivered to the site of the disease (Mazza et al. ACS Nano 7, 1016-1026 (2013); Siew et al. Mol Pharm 9, 14-28 (2012); Lalatsa et al. J Control Release 161, 523-536 (2012); Lalatsa et al. Mol Pharm 9, 1665-1680 (2012); Garrett et al. J Biophotonics 5, 458-468 (2012); Uchegbu, Expert Opin Drug Deliv 3, 629-640 (2006); Uchegbu et al. Int J Pharm 224, 185-199 (2001); Qu et al. Biomacromolecules 7, 3452-3459 (2006)).
[0121] Several types of particle delivery systems and/or formulations are known to be useful in a diverse spectrum of biomedical applications. In general, a particle is defined as a small object that behaves as a whole unit with respect to its transport and properties. Particles are further classified according to diameter. Coarse particles cover a range between 2,500 and 10,000 nanometers. Fine particles are sized between 100 and 2,500 nanometers. Ultrafine particles, or nanoparticles, are generally between 1 and 100 nanometers in size. The basis of the 100-nm limit is the fact that novel properties that differentiate particles from the bulk material typically develop at a critical length scale of under 100 nm.
[0122] As used herein, a particle delivery system/formulation is defined as any biological delivery system/formulation, which includes a particle in accordance with the present invention. A particle in accordance with the present invention is any entity having a greatest dimension (e.g. diameter) of less than 100 microns (μμm). In some embodiments, inventive particles have a greatest dimension of less than 10 p.m. In some embodiments, inventive particles have a greatest dimension of less than 2000 nanometers (nm). In some embodiments, inventive particles have a greatest dimension of less than 1000 nanometers (nm). In some embodiments, inventive particles have a greatest dimension of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. Typically, inventive particles have a greatest dimension (e.g., diameter) of 500 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 250 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 200 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 150 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 100 nm or less. Smaller particles, e.g., having a greatest dimension of 50 nm or less are used in some embodiments of the invention. In some embodiments, inventive particles have a greatest dimension ranging between 25 nm and 200 nm.
[0123] Particle characterization (including e.g., characterizing morphology, dimension, etc.) is done using a variety of different techniques. Common techniques are electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy, dual polarization interferometry and nuclear magnetic resonance (NMR). Characterization (dimension measurements) may be made as to native particles (i.e., preloading) or after loading of the cargo (herein cargo refers to e.g., one or more components of CRISPR-Cas system e.g., CRISPR enzyme or mRNA or guide RNA, or any combination thereof, and may include additional carriers and/or excipients) to provide particles of an optimal size for delivery for any in vitro, ex vivo and/or in vivo application of the present invention. In certain preferred embodiments, particle dimension (e.g., diameter) characterization is based on measurements using dynamic laser scattering (DLS).
[0124] Particle delivery systems within the scope of the present invention may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal particles. As such any of the delivery systems described herein may be provided as particle delivery systems within the scope of the present invention.
[0125] Despite microglia having a developmental origin distinct from that of bone marrow-derived myelomonocytes (Ginhoux et al. Science 330, 841-845 (2010), the contents of which are incorporated herein by reference in their entirety), it has recently been demonstrated that under specific experimental conditions, cells of donor origin showing a microglia-like phenotype and expressing some microglia surface markers could be successfully generated in the brain of mice transplanted with donor HSPCs. HSPCs have the capacity to generate new populations of myeloid and microglia cells that can exert therapeutic effects in the central nervous system (CNS). This disclosure provides compositions comprising cells modified to have a transgene comprising a truncated TSPO promoter and enhanced methods for engrafting such cells.
[0126] Transplantating HSPCs or their progeny generates transcriptionally-dependable microglia through a stepwise process similar to physiological post-natal microglia maturation. Cells comprising a transgene having a truncated TSPO promoter are able to generate new microglia upon transplantation into myeloablated recipients are retained within human and murine long-term hematopoietic stem cells (HSCs). In some embodiments, microglia cells can be generated after intracerebroventricular delivery of cells (e.g., HSPCs) comprising a transgene having a truncated TSPO promoter, which unexpectedly results in faster and more widespread microglia replacement compared to delivery of wild-type cells.
Pharmaceutical Compositions
[0127] Compositions contemplated in the present disclosure include pharmaceutical compositions comprising cells that are modified to have a transgene comprising a truncated TSPO promoter. In some embodiments, the transgene encodes a therapeutic polypeptide, and the truncated TSPO promoter drives expression of the polypeptide. In some embodiments, the therapeutic polypeptide is neuroprotective or is required to replace a missing metabolic enzyme. The pharmaceutical compositions contemplated herein can comprise autogenic or allogenic cells. In some embodiments the cells are HSPCs comprising a transgene having a truncated TSPO promoter.
[0128] The cells comprising a transgene having a truncated TSPO promoter as described herein can be administered as therapeutic compositions (e.g., as pharmaceutical compositions). Cellular compositions as described herein can be provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. A liquid preparation may be easier to prepare than a gel, another viscous composition, and a solid composition. Additionally, a liquid composition may be more convenient to administer (i.e., by injection). Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise a carrier, which can be a solvent or dispersing medium comprising, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.
[0129] Sterile injectable solutions can be prepared by incorporating the cells described herein in a sufficient amount of an appropriate diluent. Such compositions may be in admixture with a suitable carrier or excipient such as sterile water, physiological saline, glucose, dextrose, or another carrier or excipient suitable for delivering live cells to a subject. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “Remington's Pharmaceutical Science”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
[0130] Additives that enhance the stability and sterility of the cellular compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by an antibacterial or antifungal agent including, but not limited to, parabens, chlorobutanol, phenol, and sorbic acid. According to the present disclosure, however, any vehicle, diluent, or additive used must be compatible with the cells.
[0131] The compositions can be isotonic, i.e., they have the same osmotic pressure as blood and cerebrospinal fluid. The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride may be suitable for buffers containing sodium ions.
[0132] Viscosity of the compositions, if desired, can be maintained at a selected level using a pharmaceutically acceptable thickening agent. In some embodiments, the thickening agent is methylcellulose, which is readily and economically available and is easy to work with. Other suitable thickening agents include, but are not limited to, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, and carbomer. The concentration of the thickener will depend upon the agent selected and the amount of the agent used. Suitable carriers and other additives may be chosen depending on the route of administration and the nature of the dosage form (e.g., a liquid dosage form can be formulated into a solution, a suspension, a gel, or another liquid form, such as a time release formulation or liquid-filled form).
[0133] An effective amount of cells to be administered can vary for the subject being treated. In one embodiment, between about 10.sup.4 to about 10.sup.8 cells, and in another embodiment between about 10.sup.5 to about 10.sup.7 cells are administered to a subject.
[0134] The skilled artisan can readily determine the amounts of cells comprising a transgene having a truncated TSPO promoter and optional additives, vehicles, and/or carrier in compositions to be administered. In one embodiment any additive (in addition to the cell(s)) is present in an amount of about 0.001% to about 50% (weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001% to about 5 wt %. In another embodiment, the active ingredient is present at about 0.0001% to about 1 wt %. In yet another embodiment, the active ingredient is present at about 0.0001% to about 0.05 wt %. In still other embodiments, the active ingredient is present at about 0.001% to about 20 wt %. In some embodiments, the active ingredient is present at about 0.01% to about 10 wt %. In another embodiment, the active ingredient is present at about 0.05% to about 5 wt %. For any composition to be administered to an animal or human, and for any particular method of administration, toxicity can be determined by measuring the lethal dose (LD) and LD5o in a suitable animal model e.g., a rodent such as mouse. The dosage of the composition(s), concentration of components therein, and timing of administering the composition(s), which elicit a suitable response can also be determined. Such determinations do not require undue experimentation in light of the knowledge of the skilled artisan, this disclosure, and the documents cited herein. The time for sequential administrations can also be ascertained without undue experimentation.
Methods of Treatment
[0135] The present disclosure provides methods of treatment for a subject in need thereof by administering a cell comprising a transgene having a truncated TSPO promoter operably linked to a coding sequence, or a pharmaceutical composition comprising the cell, to the subject. In some embodiments, the subject in need of treatment has or is suspected of having a metabolic or neurological disease.
[0136] A health care professional may diagnose a subject as having a metabolic or neurological disease by the assessment of one or more symptoms of disease in the subject. The present disclosure provides methods of treating a metabolic or neurological disease or symptoms thereof that comprise administering to a subject (e.g., a mammal, such as a human) a therapeutically effective amount of a cell comprising a transgene having a truncated TSPO promoter drives expression of a therapeutic polynucleotide or polypeptide. In some embodiments, the cell is an HSPC or its progeny. In some embodiments, the cell is a microglial progenitor cell. In some embodiments, the cell is a microglia cell. Thus, the method in some embodiments comprises administering to the subject a therapeutically effective amount of a cell described herein sufficient to treat a metabolic or neurological disease or symptom thereof, under such conditions that the disease is treated.
[0137] The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of cells described herein, or a composition comprising such cells as described herein to produce such effect. Such treatment will be suitably administered to a subject, particularly a human, suffering from, having, susceptible to, or at risk for, a metabolic or neurological disease, or a symptom thereof. In some embodiments, the methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect.
[0138] In some embodiments, the cell or the composition comprising the cell is administered to a subject in a targeted manner. For example, in some embodiments, a composition comprising a cell having a transgene comprising a truncated TSPO promoter is administered directly to a subject's brain. In some embodiments, the composition is delivered directly to the brain via intracerebroventricular administration. In some embodiments, the composition is delivered in this manner to the lateral ventricles of the subject's brain. Methods of administration useful in the present disclosure are described, for example in International Application No. PCT/US2020/045106, which is incorporated by reference in its entirety.
[0139] Alternatively, the composition may be delivered systemically, such as by intravenous administration. Cells administered in such a manner must traverse the blood brain barrier prior to engrafting in the subject's brain. Other modes of administration (parenteral, mucosal, implant, intraperitoneal, intradermal, transdermal, intramuscular, intracerebroventricular injection, intravenous including infusion and/or bolus injection, and subcutaneous) are generally known in the art. In some embodiments, cells are administered in a medium suitable for injection, such as phosphate buffered saline, into a subject. Because the cells being administered to the subject are intended to repopulate microglial cells, intracerebroventricular administration may be advantageous to routes that require crossing the blood brain barrier.
[0140] In some embodiments, the transplanted cells are meant to replace endogenous cells (i.e., microglial cells); therefore, methods of treating a subject having, susceptible to, or at risk of developing a metabolic or neurological disease further comprise administering to a subject an agent for ablating endogenous cells, such as microglia prior to administering a cell comprising a transgene encoding a therapeutic polypeptide and having a truncated TSPO promoter. In some embodiments, the agent is an alkylating agent. In some embodiments, the alkylating agent is busulfan. In some embodiments, nanoparticle delivery of alkylating agents may be effective in creating a suitable environment for engraftment of transplanted cells, as described in International Application No. PCT/US2017/056774, the contents of which are incorporated herein by reference in their entirety.
Kits
[0141] The present disclosure contemplates kits for the treatment or prevention of a metabolic or neurological disease. In some embodiments, the kit comprises a composition comprising a cell that has a transgene comprising a truncated TSPO promoter. In some embodiments, the cell comprising a transgene having a truncated TSPO promoter is modified to express a therapeutic polypeptide. The kit can include instructions for a treatment protocol, reagents, equipment (test tubes, reaction vessels, needles, syringes, etc.), and standards for calibrating or conducting the treatment protocol. The instructions provided in a kit according to the present disclosure may be directed to suitable operational parameters in the form of a detectable label or a separate insert. Optionally, the kit may further comprise a standard or control information so that the test sample can be compared with the control information standard to determine if a consistent result is achieved. In some embodiments, the kit includes a nanoparticle for ablative conditioning of endogenous microglial cells.
[0142] In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic cellular composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
[0143] If desired a composition of the invention is provided together with instructions for administering the composition to a subject having or at risk of developing a metabolic or neurological disease or disorder of the central nervous system. The instructions will generally include information about the use of the composition for the treatment or prevention of the disease or disorder. In other embodiments, the instructions include at least one of the following: description of the therapeutic composition; dosage schedule and administration for treatment or prevention of a neurological disease or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
[0144] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
[0145] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
EXAMPLES
Example 1: Murine TSPO Promoter Cloning and Testing: Proof of Concept Study
[0146] Cells transduced via a lentiviral vector comprising a nucleic acid molecule having the full-length mTspo promoter operably linked to a coding sequence for GFP were able to express the reporter gene. A total of 2.7 kb of genomic DNA from mouse genomic DNA containing the promoter sequence 5′ of the murine TSPO gene were PCR cloned into T2700, a third-generation lentiviral transfer plasmid. The vector contains a cassette encoding GFP and the Woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) (
[0147] One limitation for a broader use of native promoter sequences in lentiviral vectors is the cargo limitations of vector capsids. To overcome this problem, fragments of the mTspo 5′ UTR were cloned and tested for promoter activity. The following novel transfer plasmids were generated: T1600, T1200, and T300 containing approximately 1.6 kb, 1.2 kb, and 300 base pairs of the mTspo 5′ UTR, respectively (
[0148] Lentiviral vector supernatants from the HEK293T cultures were used to test the infectivity of the vectors in several cell lines: human fibroblast, HEK293T; mouse microglia, BV-2; and mouse fibroblast, NIH3T3. Ectopic GFP expression driven by the mTspo promoter in mouse fibroblasts depended greatly on the distal 1 kb in the 5′ region of the full promoter, but eliminating this terminal sequence did not have a drastic effect on GFP expression in microglia cells under basal conditions (
[0149] Using pools of transduced cells, the effect of LPS (1 μg/ml) on GFP expression was tested. A change in GFP intensity was observed only when BV-2 microglia cells were challenged with LPS, while LPS did not affect GFP expression driven by any of the mTspo vectors assayed in NIH3T3 fibroblasts (
[0150] A series of transplantations into C57/BL mice took advantage of the CD45.1-CD45.2 mismatch system (
[0151] The full length mTspo promoter, T1600, T1200, T300, and a non-inducible PGK-GFP control were tested (Table 1). To maximize responsiveness of the murine TSPO promoters, the transplanted mice in each cohort were split into two cohorts: one to assess basal GFP expression and microglia reconstitution, and the other was challenged with LPS one day before sacrifice. The longest murine TSPO promoters achieved modest levels of labelling of HSPCs (
[0152] During the active process of expansion and differentiation of the transplanted cell progeny, the majority of microglia are transiently activated (Tap), and these cells have an increased level of murine TSPO expression, which complicates evaluation of the impact of TLR4 activation. For this reason, shortly after transplant, the animals were divided into two timepoints of analysis. A small fraction of the transplanted animals at 45 days after transplant were analyzed, but the majority of the animals were evaluated at six months post-transplant.
[0153] Upon perfusion with saline, brains, bone marrow, and spleen were collected and processed for flow cytometry. Analysis confirmed induction of GFP expression in donor-derived microglia-like cells (CD45.sup.+CD11b.sup.+) cells upon LPS stimulation (
TABLE-US-00008 TABLE 1 Lin- Group donor Transduction MOI Recipient Conditioning Dose Treatment A CD45.1 full-Tspo (2700 bp) 100 CD45.2 Busulfan 4 × 25 mg/kg n/a LPS 24 h before sac B CD45.1 PGK (700 bp) 100 CD45.2 Busulfan 4 × 25 mg/kg n/a LPS 24 h before sac C CD45.1 1600 bp Tspo 100 CD45.2 Busulfan 4 × 25 mg/kg n/a LPS 24 h before sac D CD45.1 1200 bp Tspo 100 CD45.2 Busulfan 4 × 25 mg/kg n/a LPS 24 h before sac E CD45.1 300 bp Tspo 100 CD45.2 Busulfan 4 × 25 mg/kg n/a LPS 24 h before sac
Example 2: Human TSPO Promoter Cloning and Testing for Clinical Development
[0154] Based on the positive findings observed using the murine TSPO promoter sequences in a homologous setting, use of the human TSPO promoter sequence to drive microglia specific and ambient responsive expression of therapeutic transgenes was investigated. Despite the large amount of human genomic information and the extensive use of TSPO radioligands to follow patients undergoing neurodegenerative disease progression, the characterization of the human promoter driving TSPO expression has not been thoroughly addressed. Mouse and human TSPO sequences are in genomic loci with very poor homology, and because the human TSPO putative promoter region extends over several kilobases on chromosome 22, it is not suitable to use as such in a lentiviral vector for comparative analyses. Hence, synthetic promoters derived from the human sequence were generated that were bioinformatically predicted to control human TSPO expression. Special interest was directed to those synthetic promoters regulating TSPO expression in microglia-related tissues found in the ENCODE and other databases (
[0155] To conserve as much of the genuine specificity established by the union of transcription factors, while keeping a reduced size suitable for use in viral vectors, the following promoter sequences were cloned: [0156] i) The P1 5′ promoter [0157] ii) The extended P1+P2 promoter [0158] iii) The synthetic concatenation of P1, P2 and the intronic enhancer E2 [0159] iv) The synthetic union of E1+P1+P2+E2
These promoter sequences were cloned into the same lentiviral vector transfer plasmid employed for murine TSPO (
[0160] Constructs are selected for further expression characterization using LPS or other TLR-4 agonist responsiveness in relevant cell lines. Additionally, in vivo characterization of the most promising constructs is studied in a humanized model of microglia reconstitution in NSG animals. In vivo exploratory assessment of responsiveness of the promoter(s) in a mouse-into-mouse setting can be assessed.
Sequence of lentiviral vector:
TABLE-US-00009 pCCLsin.PPT.hPGK.GFP.Wpre_mut_AMP caggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgata aatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcac ccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttg agagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagca actcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaatt atgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcaca acatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtag caatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagtt gcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagc actggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctga gataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatcta ggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatctt cttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaact ctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtag caccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagtt accggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagataccta cagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcg cacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtca ggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatc ccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcga ggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactgga aagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgg aattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctg gagctgcaagcttggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattatt gactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggct gaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggag tatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctgg cattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtaca tcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacggg actttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtg aaccggggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttga gtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgc ccgaacagggacctgaaagcgaaagggaaaccagagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggg gcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggaga attagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaa cgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaag aacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagagg aagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtg aattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcag tgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcctcaatgacgctgacggtacaggccagacaatt attgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagc tccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgct gtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacac aagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaa ttggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagt gaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaag aaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattgggg ggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatc gatcacgagactagcctcgagaagcttgatatcgaattcccacggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggacg cggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttc gccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaa acggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaat agcggctgctcagcggggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgt tcctgcccgcgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctcccca gggggatccaccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgta aacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagc tgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttctt caagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcg agggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaacta caacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagc gtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccg ccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctg tacaagtaaagcggccgcgtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgt ggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagtt gtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttc cgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactg acaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtc ccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcg gatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaa agaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagc ctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgact ctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaag aaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattttttt cactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccagttccgcccattct ccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggag gcctaggcttttgcgtcgagacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactggga aaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcc caacagttgcgcagcctgaatggcgaatggcgcgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtga ccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgg gggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgat agacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattctttt gatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaat ttcc
Example 3: Design of Novel Promoter/Enhancer Elements Based on the Human Translocator Protein (TSPO) Putative Regulatory Region
[0161] In the genomic region around the human translocator protein (TSPO) Transcriptional Start Site (TSS) on chromosome 22, four clusters of putative transcriptional regulatory elements of the human TSPO gene were identified (i.e., P1, P2, E1, and E2) by integrating publicly-available transcriptional and epigenetic data produced by the Broad Institute-Encode Project using human hematopoietic K652 cells. In particular, regions were identified that were epigenetically marked as active promoters (H3K4me3.sup.+ and H3K27ac.sup.+) or active enhancers (H3K4me1.sup.+ and H3K27ac.sup.+) bound by hematopoietic-specific transcription factors as detected by Chip-seq genomic maps (
[0162] In summary, two enhancer elements (E1 and E2) and two promotorial regions (P1 and P2) were identified. P2 and E2 were located downstream to the translocator protein (TSPO) transcriptional start site (TSS), within the TSPO gene body, therefore representing a transcribed portion of the translocator protein (TSPO) core promoter (P2) and a putative intragenic enhancer (E2), respectively.
Example 4: Production of Lentiviral Vectors Carrying Translocator Protein-Based (TSPO-Based) Promoter/Enhancer Elements to Drive Green Fluorescent Protein (GFP) Expression
[0163] In order to make these putative regulatory elements suitable for cloning into a lentiviral vector system, genomic sequences were modified extensively to eliminate all the canonical and cryptic signals of poly-adenylation, which could lead to premature stop of the lentiviral genome production in the packaging cells, and signals of splicing that could induce transcriptional alterations in the transduced cells by trans-splicing events occurring between the integrated provirus and the host genome. After sequence editing, the four putative regulatory regions (P1, P2, El, and E1) derived from the human translocator protein (TSPO) genomic locus were combined into five regulatory cassettes that were then cloned into five corresponding lentiviral vectors (LVs) to drive the expression of a green fluorescent protein (GFP) reporter gene, see
[0164] The five lentiviral vectors (LVs) were produced in third-generation lentiviral vectors and pseudotyped with the Vesicular Stomatitis Virus (VSV) g-protein. The resulting viral infectious titers ranged from 5×10.sup.8 to 1×10.sup.9 Transducing Units (TU)/mL. Although the different lentiviral vectors (LVs) showed a variable infectivity in human microglial cell lines HMC3, they were all able to efficiently transduce relevant target cells (
Example 5: Evaluation of Gene Expression Driven by the Translocator Protein-Based (TSPO-Based) Promoter/Enhancer Elements In Vitro
[0165] To test the capability of the five regulatory cassettes to drive the green fluorescent protein (GFP) expression in a similar fashion to the endogenous translocator protein (TSPO) promoter, human microglial HMC3 cells were transduced with the five lentiviral vectors (LVs) and green fluorescent protein (GFP) mRNA and protein expression were analyzed before and after microglial cell-activation.
[0166] In vivo, microglia activation is triggered by a plethora of well described subsets of immune receptors such as Toll-like receptors (TLRs), scavenger receptors, and by several cytokine and chemokine receptors. In vitro, HMC3 microglial cells can be activated by incubation with Interferon (IFN)-γ.
[0167] The best results were obtained incubating the cells for 72 hours with 100 ng/ml of IFN-γ (
[0168] In the HMC3 cells transduced with the different lentiviral vectors (LVs), a concomitant and significant increase of major histocompatibility complex II (MHC-II), translocator protein (TSPO), and green fluorescent protein (GFP) expression was observed upon cell-activation, see
[0169] To precisely evaluate transcriptional regulation driven by the five translocator protein-based (TSPO-based) regulatory cassettes relative to endogenous translocator protein (TSPO) promoter activity, the mRNA level of the lentiviral green fluorescent protein (GFP) and the endogenous translocator protein (TSPO) was quantified and correlated before and after microglial cell-activation. If the lentiviral cassettes were able to closely reproduce the transcriptional regulation operated by the endogenous translocator protein (TSPO) promoter, a similar increase of mRNA expression was observed for the green fluorescent protein (GFP) and the translocator protein (TSPO) transcripts.
[0170] HMC3 cells mock-transduced or lentiviral vector-transduced (LV-transduced) cells were activated or not with the IFN-γ. Then, total mRNA was collected, reverse-transcribed and analyzed by real-time polymerase chain reaction (real-time PCR) (with digital-droplet polymerase chain reaction (PCR) technology). Specific primers were designed to quantify the mRNA level of human translocator protein (TSPO), lentiviral green fluorescent protein (GFP) (on the viral Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) region), and major histocompatibility complex II (MHC-II) (as an additional control) in the same reaction. The resulting total mRNA amounts of the specific genes was normalized to the GAPDH (glyceraldenyde 3-phosphate dehydrogenase) gene and for the specific vector copy number (VCN) in the transduced cells in order to infer the level of transgene expression driven by a single copy of each lentiviral vector (LV) (green fluorescent protein (GFP)/copy).
[0171] As shown in
Summary of Examples 3 to 5
[0172] In vitro data produced in human microglial cell lines for the transcriptional activity of novel translocator protein (TSPO)-based regulatory cassettes, both at the protein and mRNA level, supported efficacy of at least the MPPO1 (034) and MMPO7 (037) lentiviral vector (LV) regulatory cassettes in human relevant cells in vivo to safely and specifically induce a therapeutic level of transgene expression in pathological conditions of the central nervous system (CNS), as in the presence of neuroinflammation.
Other Embodiments
[0173] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
[0174] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
[0175] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. This application may be related to PCT/US2020/045106 and PCT/US2017/05677, as well as the PCT applications, entitled “COMPOSITIONS AND METHODS FOR TREATING AMYOTROPHIC LATERAL SCLEROSIS” and “COMPOSITIONS AND METHODS FOR TREATING ALZHEIMER′S DISEASE”, each filed Oct. 1, 2020, which claim priority to the following provisional applications, respectively, 62/908,942 and 62/908,913, all of which are incorporated herein by reference.