Genomic safe harbors for genetic therapies in human stem cells and engineered nanoparticles to provide targeted genetic therapies

1. A method of genetically modifying a cell through homology-directed repair (HDR) within a genomic safe harbor loci wherein the method comprises:

contacting the cell with a gold nanoparticle (AuNP) comprising at least two active layers wherein the first layer comprises

a crRNA with a 3′ end and a 5′ end,

wherein the 3′ end is conjugated to a spacer with a thiol modification, and the 5′ end is conjugated to a nuclease to form a crRNA-nuclease ribonucleoprotein (RNP) complex, and

wherein the thiol modification is covalently linked to the surface of a gold core of the AuNP

and wherein the second layer comprises

a donor template comprising a therapeutic gene and HDR templates and wherein the second layer is farther from the surface of the gold core than the first layer and

wherein the crRNA-nuclease RNP complex binds a PAM-site within human (h) chromosome (chr) 11 at positions 67681215-67741765, 67691162-67691186, 67723825-67723849, 67805337-67845629, 67812349-67812375, 67812443-67812469, 67839126-67839150, or 67895738-67941098 or hchr3 at positions 46373915-46373939 and results in cutting of the DNA at the PAM of the PAM-site

thereby genetically modifying the cell through HDR within the genomic safe harbor loci.

2. The method of claim 1 , wherein the cell is a hematopoietic stem cell (HSC), a hematopoietic progenitor cell (HPC), a hematopoietic stem and progenitor cell (HSPC), a T cell, a natural killer (NK) cell, a B cell, a macrophage, a monocyte, a mesenchymal stem cell (MSC), a white blood cell (WBC), a mononuclear cell (MNC), a endothelial cell (EC), a stromal cell, and/or a bone marrow fibroblast.

3. A nanoparticle (NP) comprising a metallic core associated with two active NP layers wherein the first NP layer comprises

a crRNA with a 3′ end and a 5′ end,

wherein the 3′ end is conjugated to a spacer with a thiol modification, and the 5′ end is conjugated to a nuclease to form a crRNA-nuclease ribonucleoprotein (RNP) complex,

wherein the thiol modification is covalently linked to the surface of the metallic NP core and

wherein the crRNA-nuclease RNP complex binds a target PAM-site within human (h) chromosome (chr) 11 at positions 67681215-67741765; 67691162-67691186; 67723825-67723849; 67805337-67845629; 67812349-67812375; 67812443-67812469; 67839126-67839150; or 67895738-67941098 or hchr3 at positions 46373915-46373939 and results in cutting of the DNA at the PAM of the PAM-site;

and wherein the second NP layer comprises a donor template comprising a therapeutic gene and homology-directed repair templates; and wherein the second NP layer is farther from the surface of the metallic core than the first NP layer.

4. The NP of claim 3 , wherein the metallic core is less than 20 nm in diameter.

5. The NP of claim 3 , wherein the metallic core is gold.

6. The NP of claim 3 , further comprising a positively-charged polymer coating.

7. The NP of claim 6 , wherein the positively-charged polymer coating surrounds the RNP complex and contacts the surface of the metallic core.

8. The NP of claim 6 , wherein the positively-charged polymer has a molecular weight of less than 2500 daltons.

9. The NP of claim 6 , wherein the positively-charged polymer coating comprises polyethyleneimine with a molecular weight of 2000 daltons.

10. The NP of claim 3 , wherein

the target site is hchr11: 67723825-67723849 and has the sequence as set forth in SEQ ID NO: 132,

the target site is hchr11:67691162-67691186 and has the sequence as set forth in SEQ ID NO: 108,

the target site is hchr11:67812349-67812375 and has the sequence as set forth in SEQ ID NO: 203,

the target site is hchr11:67839126-67839150 and has the sequence as set forth in SEQ ID NO: 210, or

the target site is hchr3:46373915-46373939 and has the sequence as set forth in SEQ ID NO: 212.

11. The NP of claim 3 , wherein the crRNA comprises:

SEQ ID NO: 195 or a sequence with at least 98% identity to SEQ ID NO: 195 that binds within Hchr11: 67723825-67723849;

SEQ ID NO: 196 or a sequence with at least 98% identity to SEQ ID NO: 196 that binds within Hchr11: 67691162-67691186,

SEQ ID NO: 209 or a sequence with at least 98% identity to SEQ ID NO: 209 that binds within Hchr11: 67812349-67812375,

SEQ ID NO: 211 or a sequence with at least 98% identity to SEQ ID NO: 211 that binds within hchr11:67839126-67839150, or

SEQ ID NO: 260 or a sequence with at least 98% identity to SEQ ID NO: 260 that binds within hchr3:46373915-46373939.

12. The NP of claim 3 , wherein the nuclease is Cpf1 or Cas9.

13. The NP of claim 3 , wherein the nuclease is Cpf1 and has a sequence as set forth in SEQ ID NOs: 229-241 or is encoded by a sequence as set forth in SEQ ID NOs: 216-227.

14. The NP of claim 3 , wherein the NP is coupled to a targeting molecule through a linkage with the nuclease.

15. The NP of claim 14 , wherein the targeting molecule comprises a CD34 binding domain or a CD90 binding domain.

16. The NP of claim 3 , wherein the therapeutic gene comprises or encodes skeletal protein 4.1, glycophorin, p55, the Duffy allele, globin family genes; WAS; phox; dystrophin; pyruvate kinase; CLN3; ABCD1; arylsulfatase A; SFTPB; SFTPC; NLX2.1; ABCA3; GATA1; ribosomal protein genes; TERT; TERC; DKC1; TINF2; CFTR; LRRK2; PARK2; PARK7; PINK1; SNCA; PSEN1; PSEN2; APP; SOD1; TDP43; FUS; ubiquilin 2; C9ORF72, α2β1; αvβ3; αvβ5; αvβ63; BOB/GPR15; Bonzo/STRL-33/TYMSTR; CCR2; CCR3; CCRS; CCR8; CD4; CD46; CD55; CXCR4; aminopeptidase-N; HHV-7; ICAM; ICAM-1; PRR2/HveB; HveA; α-dystroglycan; LDLR/α2MR/LRP; PVR; PRR1/HveC, laminin receptor, 101F6, 123F2, 53BP2, abl, ABLI, ADP, aFGF, APC, ApoAl, ApoAlV, ApoE, ATM, BAI-1, BDNF, Beta*(BLU), bFGF, BLC1, BLC6, BRCA1, BRCA2, CBFA1, CBL, C-CAM, CFTR, CNTF, COX-1, CSFIR, CTS-1, cytosine deaminase, DBCCR-1, DCC, Dp, DPC-4, E1A, E2F, EBRB2, erb, ERBA, ERBB, ETS1, ETS2, ETV6, Fab, FancA, FancB, FancC, FancD1, FancD2, FancE, FancF, FancG, Fancl, FancJ, FancL, FancM, FancN, FancO, FancP, FancQ, FancR, FancS, FancT, FancU, FancV, and FancW, FCC, FGF, FGR, FHIT, fms, FOX, FUS 1, FUS1, FYN, G-CSF, GDAIF, Gene 21, Gene 26, GM-CSF, GMF, gsp, HCR, HIC-1, HRAS, hst, IGF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, ING1, interferon α, interferon β, interferon y, IRF-1, JUN, KRAS, LCK, LUCA-1, LUCA-2, LYN, MADH4, MADR2, MCC, mda7, MDM2, MEN-I, MEN-II, MLL, MMAC1, MYB, MYC, MYCL1, MYCN, neu, NF-1, NF-2, NGF, NOEY1, NOEY2, NRAS, NT3, NTS, OVCA1, p16, p21, p27, p53, p57, p73, p300, PGS, PIM1, PL6, PML, PTEN, raf, Rap1A, ras, Rb, RB1, RET, rks-3, ScFv, scFV ras, SEM A3, SRC, TALI, TCL3, TFPI, thrombospondin, thymidine kinase, TNF, TP53, trk, T-VEC, VEGF, VHL, WT1, WT-1, YES, zac1, iduronidase, IDS, GNS, HGSNAT, SGSH, NAGLU, GUSB, GALNS, GLB1, ARSB, HYAL1, F8, F9, HBB, CYB5R3, γC, JAK3, IL7RA, RAG1, RAG2, DCLRE1C, PRKDC, LIG4, NHEJ1, CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI1, STIM1, CORO1A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1, TERT, TINF2, DCLRE1B, and SLC46A1.

17. A cell genetically-modifed by a method of claim 1 .

18. The cell of claim 17 , wherein the cell is a hematopoietic stem cell (HSC), a hematopoietic progenitor cell (HPC), a hematopoietic stem and progenitor cell (HSPC), a T cell, a natural killer (NK) cell, a B cell, a macrophage, a monocyte, a mesenchymal stem cell (MSC), a white blood cell (WBC), a mononuclear cell (MNC), a endothelial cell (EC), a stromal cell, and/or a bone marrow fibroblast.

19. The cell of claim 17 , wherein the cell is a CD34 + CD45RA − CD90 + HSC.

20. The cell of claim 17 , wherein the cell is a human blood cell.