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1. WO2020118110 - REDUCED AND MINIMAL MANIPULATION MANUFACTURING OF GENETICALLY-MODIFIED CELLS

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[ EN ]

CLAIMS

What is claimed is:

1. A method of genetically modifying a hematopoietic stem and progenitor cell (HSPC) population in a biological sample comprising adding a gold nanoparticle (AuNP) to the biological sample, wherein the AuNP comprises

a gold (Au) core that is less than 20 nm in diameter;

a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) guide RNA (crRNA)-nuclease ribonucleoprotein (RNP) complex wherein the crRNA comprises 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 the nuclease, and wherein the thiol modification is covalently linked to the surface of the Au core and wherein the crRNA has a sequence set forth in SEQ ID NO: 262; SEQ ID NO: 13; SEQ ID NO: 14; or SEQ ID NO: 241-261 ;

a positively-charged polyethyleneimine polymer coating wherein the positively-charged polyethyleneimine polymer has a molecular weight of less than 2500 daltons, surrounds the RNP complex, and contacts the surface of the Au core; and

a donor template comprising a homology-directed repair template (HDT) on the surface of the positively-charged polymer coating wherein the HDT template comprises a sequence set forth in SEQ ID NO: 48; SEQ ID NO: 4; SEQ ID NO: 15; SEQ ID NO: 33-41 ; SEQ ID NO: 44- 47; or SEQ ID NO: 49-51 ; and

a CD133 targeting ligand comprising a binding domain of antibody clone REA820, REA753, REA816, 293C3, AC141 , AC133, or 7

wherein the targeting ligand is linked to the nuclease through an amine-to-sulfhydryl crosslinker or a sulfhydryl-to-sulfhydryl crosslinker and

wherein the HSPC population has not been exposed to electroporation, a viral vector encoding an HDT, or a magnetic cell separation process, and wherein the method results in no more than 30% HSPC cellular toxicity and provides a gene-editing efficiency within the HSPC population of at least 10%.

2. The method of claim 1 , wherein the crRNA targets a sequence set forth in SEQ ID NO: 25;

SEQ ID NO: 3; SEQ ID NO: 24; SEQ ID NO: 26- 32; SEQ ID NO: 42; SEQ ID NO: 43; or SEQ ID NO: 214-224.

3. The method of claim 1 , wherein the crRNA has a sequence as set forth in SEQ ID NO: 262, SEQ ID NO: 261 or SEQ ID NO: 259.

4. The method of claim 1 , wherein the nuclease comprises Cpf1 or Cas9.

5. The method of claim 1 , wherein the positively-charged polymer coating comprises

polyethyleneimine with a molecular weight of 2000 daltons.

6. The method of claim 1 , wherein the weight/weight (w/w) ratio of Au core to nuclease is 0.6.

7. The method of claim 1 , wherein the w/w ratio of Au core to HDT is 1.0.

8. A method of genetically modifying a selected cell population in a biological sample comprising adding a gold nanoparticle (AuNP) to the biological sample, wherein the AuNP comprises a gold (Au) core that is less than 30 nm in diameter;

a guide RNA (gRNA)-nuclease ribonucleoprotein (RNP) complex wherein the gRNA comprises a 3’ end and a 5’ end, wherein the 3’ end is conjugated to a spacer with a chemical modification, and the 5’ end is conjugated to the nuclease, and wherein the chemical modification is covalently linked to the surface of the Au core;

a positively-charged polymer coating wherein the positively-charged polymer has a molecular weight of less than 2500 daltons, surrounds the RNP complex, and contacts the surface of the Au core; and

a donor template comprising a homology-directed repair template (HDT) on the surface of the positively-charged polymer coating

wherein the selected cell population has not been exposed to electroporation or a viral vector encoding an and wherein the method results in no more 30% cellular toxicity of the selected cell population and provides a gene-editing efficiency within the selected cell population of at least

10%.

9. The method of claim 8, wherein the weight/weight (w/w) ratio of Au core to nuclease is 0.6.

10. The method of claim 8, wherein the w/w ratio of Au core to HDT is 1.0.

11. The method of claim 8, wherein the AuNP is less than 70 nm in diameter.

12. The method of claim 8, wherein the AuNP has a polydispersity index (PDI) of less than 0.2.

13. The method of claim 8, wherein the gRNA comprises a Clustered Regularly Interspaced Short

Palindromic Repeat (CRISPR) crRNA.

14. The method of claim 13, wherein the crRNA targets a sequence as set forth in SEQ ID NO:

1 ; SEQ ID NO: 3; SEQ ID NO: 20 - 32; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 84 - 97; or SEQ ID NO: 214-224.

15. The method of claim 13, wherein the crRNA comprises a sequence set forth in SEQ ID NO:

5; SEQ ID NO: 6; SEQ ID NO: 13; SEQ ID NO: 14; or SEQ ID NO: 225 - 264.

16. The method of claim 8, wherein the nuclease comprises Cpf1 or Cas9.

17. The method of claim 8, wherein the positively-charged polymer coating comprises polyethyleneimine (PEI), polyamidoamine (PAMAM); polylysine (PLL), polyarginine; cellulose, dextran, spermine, spermidine, or poly(vinylbenzyl trialkyl ammonium).

18. The method of claim 8, wherein the positively-charged polymer has a molecular weight of 1500 - 2500 daltons.

19. The method of claim 8, wherein the positively-charged polymer has a molecular weight of 2000 daltons.

20. The method of claim 8, wherein the chemical modification comprises a free thiol, amine, or carboxylate functional group.

21. The method of claim 8, wherein the spacer comprises an oligoethylene glycol spacer.

22. The method of claim 21 , wherein the oligoethylene glycol spacer comprises an 18 atom oligoethylene glycol spacer.

23. The method of claim 8, wherein the HDT comprises sequences having homology to genomic sequences undergoing modification.

24. The method of claim 23, wherein the HDT comprises a sequence as set forth in SEQ ID NO:

2; SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NO: 33 - 41 ; or SEQ ID NO: 44 - 52.

25. The method of claim 8, wherein the HDT comprises single-stranded DNA (ssDNA).

26. The method of claim 8, wherein the donor template comprises a therapeutic gene.

27. The method of claim 26, 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; C90RF72, a2b1 ; anb3; anb5; anb63; BOB/GPR15; Bonzo/STRL-33/TYMSTR; CCR2; CCR3; CCR5; CCR8; CD4; CD46; CD55; CXCR4; aminopeptidase-N; HHV-7; ICAM; ICAM-1 ; PRR2/HveB; HveA; a-dystroglycan; LDLR/02MR/LRP; PVR; PRR1/HveC, laminin receptor, 101 F6, 123F2, 53BP2, abl, ABLI, ADP, aFGF, APC, ApoAI, ApoAIV, 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, FancDI , 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 a, interferon b, interferon g, 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, NT5, 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, TAL1 , TCL3, TFPI, thrombospondin, thymidine kinase, TNF, TP53, trk, T-VEC, VEGF, VHL, WT1 , WT-1 , YES, zad , iduronidase, IDS, GNS, HGSNAT, SGSH, NAGLU, GUSB, GALNS, GLB1 , ARSB, HYAL1 , F8, F9, HBB, CYB5R3, yC, JAK3, IL7RA, RAG1 , RAG2, DCLRE1 C, PRKDC, LIG4, NHEJ1 , CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI 1 , STIM1 , C0R01A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1 , TERT, TINF2, DCLRE1 B, and SLC46A1.

28. The method of claim 8, wherein the AuNP further comprises a targeting ligand linked to the nuclease.

29. The method of claim 28, wherein the AuNP with the linked targeting ligand is 60-150 nm in diameter.

30. The method of claim 28, wherein the targeting ligand comprises a binding molecule that binds CD3, CD4, CD34, CD46, CD90, CD133, CD164, a luteinizing hormone-releasing hormone (LHRH) receptor, or an aryl hydrocarbon receptor (AHR).

31. The method of claim 28, wherein the targeting ligand comprises an anti-human CD3 antibody or antigen binding fragment thereof, an anti-human CD4 antibody or antigen binding fragment thereof, an anti-human CD34 antibody or antigen binding fragment thereof, an anti-human CD46 antibody or antigen binding fragment thereof, an anti-human CD90 antibody or antigen binding fragment thereof, an anti-human CD133 antibody or antigen binding fragment thereof, an anti-human CD164 antibody or antigen binding fragment thereof, an anti-human CD133 aptamer, a human luteinizing hormone, a human chorionic gonadotropin, degerelix acetate, or StemRegenin 1.

32. The method of claim 28, wherein the targeting ligand comprises antibody clone: 581 ; antibody clone: 561 ; antibody clone: REA1 164; antibody clone: AC136; antibody clone: 5E10; antibody clone: DG3; antibody clone: REA897; antibody clone: REA820; antibody clone: REA753; antibody clone: REA816; antibody clone: 293C3; antibody clone: AC141 ; antibody clone: AC133; antibody clone: 7; aptamer A15; aptamer B19; HCG (Protein/Ligand); or Luteinizing hormone (LH Protein/Ligand).

33. The method of claim 28, wherein the nuclease and targeting ligand are linked through an amino acid linker.

34. The method of claim 33, wherein the amino acid linker comprises a direct amino acid linker, a flexible amino acid linker, or a tag-based amino acid linker.

35. The method of claim 28, wherein the nuclease and targeting ligand are linked through

polyethylene glycol (PEG).

36. The method of claim 28, wherein the nuclease and targeting ligand are linked through an amine-to-sulfhydryl crosslinker or a or sulfhydryl to sulfhydryl crosslinker.

37. The method of claim 28, wherein the nuclease and targeting ligand are linked through PEG and an amine-to-sulfhydryl crosslinker or are linked through PEG and a sulfhydryl to sulfhydryl crosslinker.

38. The method of claim 28, wherein the selected cell population has not undergone a magnetic separation process to remove the selected cells from the biological sample.

39. The method of claim 8, wherein the selected cell population comprises a blood cell selected from 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), an endothelial cell (EC), a stromal cell, and/or a bone marrow fibroblast.

40. The method of claim 39, wherein the blood cell comprises a CD34+CD45RA CD90+ HSC; a CD347CD133+ HSC; an LH+ HSC; a CD34+CD90+ HSPC; a CD34+CD90+ CD133+ HSPC; and/or an AHR+ HSPC.

41. The method of claim 39, wherein the blood cell comprises a CD3+ T cell and/or a CD4+ T cell.

42. The method of claim 8, wherein the biological sample comprises peripheral blood, bone marrow, granulocyte colony stimulating factor (GCSF) mobilized peripheral blood, and/or plerixafor mobilized peripheral blood.

43. The method of claim 8, wherein the adding is in an amount of 1 , 2, 3, 4, 5, 8, 10, 12, 15, or 20 pg of AuNP per milliliter (ml_) of biological sample.

44. The method of claim 42, wherein the biological sample and the added AuNP are incubated for 1-48 hours.

45. The method of claim 42, wherein the biological sample and the added AuNP are incubated until testing confirms the uptake of the AuNP into cells.

46. The method of claim 45, wherein the testing comprises confocal microscopy imaging, inductively coupled plasma (ICP)-mass spectrometry (ICP-MS), ICP-atomic emission spectroscopy (ICP-AES), or ICP-optical emission spectroscopy (ICP-OES).

47. A cell modified according to a method of claim 8.

48. A therapeutic formulation comprising a cell of claim 47.

49. A method of providing a therapeutic nucleic acid sequence to a subject in need thereof comprising administering a cell of claim 47 or a therapeutic formulation of claim 48 to the subject thereby providing a therapeutic nucleic acid sequence to the subject.

50. A gold nanoparticle (AuNP) comprising

a gold (Au) core that is less than 30 nm in diameter;

a guide RNA -nuclease ribonucleoprotein (RNP) complex wherein the gRNA comprises a 3’ end and a 5’ end, wherein the 3’ end is conjugated to a spacer with a chemical modification, and the 5’ end is conjugated to the nuclease, and wherein the chemical modification is covalently linked to the surface of the Au core;

a positively-charged polymer coating wherein the positively-charged polymer has a molecular weight of less than 2500 daltons, surrounds the RNP complex, and contacts the surface of the Au core; and

a donor template comprising a homology-directed repair template (HDT) on the surface of the positively-charged polymer coating.

51. The AuNP of claim 50, wherein the weight/weight (w/w) ratio of Au core to nuclease is 0.6.

52. The AuNP of claim 50, wherein the w/w ratio of Au core to HDT is 1.0.

53. The AuNP of claim 50, wherein the AuNP is less than 70 nm in diameter.

54. The AuNP of claim 50, wherein the AuNP has a polydispersity index (PDI) of less than 0.2.

55. The AuNP of claim 50, wherein the gRNA comprises a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) crRNA.

56. The AuNP of claim 55, wherein the crRNA targets a sequence as set forth in SEQ ID NO: 1 ;

SEQ ID NO: 3; SEQ ID NO: 20 - 32; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 84 - 97; or SEQ ID NO: 214-224.

57. The AuNP of claim 55, wherein the crRNA comprises a sequence as set forth in SEQ ID NO:

5; SEQ ID NO: 6; SEQ ID NO: 13; SEQ ID NO: 14; or SEQ ID NO: 225 - 264.

58. The AuNP of claim 50, wherein the nuclease comprises Cpf1 or Cas9.

59. The AuNP of claim 50, wherein the positively-charged polymer coating comprises polyethyleneimine (PEI), polyamidoamine (PAMAM); polylysine (PLL), polyarginine; cellulose, dextran, spermine, spermidine, or poly(vinylbenzyl trialkyl ammonium).

60. The AuNP of claim 50, wherein the positively-charged polymer has a molecular weight of 1500 - 2500 daltons.

61. The AuNP of claim 50, wherein the positively-charged polymer has a molecular weight of 2000 daltons.

62. The AuNP of claim 50, wherein the chemical modification comprises a free thiol, amine, or carboxylate functional group.

63. The AuNP of claim 50, wherein the spacer comprises an oligoethylene glycol spacer.

64. The AuNP of claim 63, wherein the oligoethylene glycol spacer comprises an 18 atom

oligoethylene glycol spacer.

65. The AuNP of claim 50, wherein the HDT comprises sequences having homology to genomic sequences undergoing modification.

66. The AuNP of claim 65, wherein the HDT comprises a sequence set forth in SEQ ID NO: 2;

SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NO: 33 - 41 ; or SEQ ID NO: 44 -52.

67. The AuNP of claim 50, wherein the HDT comprises single-stranded DNA (ssDNA).

68. The AuNP of claim 50, wherein the donor template comprises a therapeutic gene.

69. The AuNP of claim 68, wherein the therapeutic gene 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; C90RF72, a2b1 ; anb3; anb5; anb63; BOB/GPR15; Bonzo/STRL-33/TYMSTR; CCR2; CCR3; CCR5; CCR8; CD4; CD46; CD55; CXCR4; aminopeptidase-N; HHV-7; ICAM; ICAM-1 ; PRR2/HveB; HveA; a-dystroglycan; LDLR/02MR/LRP; PVR; PRR1/HveC, laminin receptor, 101 F6, 123F2, 53BP2, abl, ABLI, ADP, aFGF, APC, ApoAI, ApoAIV, 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, FancDI , 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-1 1 IL-12, ING1 , interferon a, interferon b, interferon g, 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, NT5, 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, TAL1 , TCL3, TFPI, thrombospondin, thymidine kinase, TNF, TP53, trk, T-VEC, VEGF, VHL, WT1 , WT-1 , YES, zad , iduronidase, IDS, GNS, HGSNAT, SGSH, NAGLU, GUSB, GALNS, GLB1 , ARSB, HYAL1 , F8, F9, HBB, CYB5R3, yC, JAK3, IL7RA, RAG1 , RAG2, DCLRE1 C, PRKDC, LIG4, NHEJ1 , CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI 1 , STIM1 , COR01A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1 , TERT, TINF2, DCLRE1 B, and SLC46A1.

70. The AuNP of claim 50, wherein the AuNP further comprises a targeting ligand linked to the

nuclease.

71. The AuNP of claim 70, wherein the targeting ligand comprises a binding molecule that binds CD3, CD4, CD34, CD46, CD90, CD133, CD164, a luteinizing hormone-releasing hormone (LHRH) receptor, or an aryl hydrocarbon receptor (AHR).

72. The AuNP of claim 70, wherein the targeting ligand comprises an anti-human CD3 antibody or antigen binding fragment thereof, an anti-human CD4 antibody or antigen binding fragment thereof, an anti-human CD34 antibody or antigen binding fragment thereof, an anti-human CD46 antibody or antigen binding fragment thereof, an anti-human CD90 antibody or antigen binding fragment thereof, an anti-human CD133 antibody or antigen binding fragment thereof, an anti-human CD164 antibody or antigen binding fragment thereof, an anti-human CD133 aptamer, a human luteinizing hormone, a human chorionic gonadotropin, degerelix acetate, or StemRegenin 1.

73. The AuNP of claim 70, wherein the targeting ligand comprises antibody clone: 581 ; antibody clone: 561 ; antibody clone: REA1 164; antibody clone: AC136; antibody clone: 5E10; antibody clone: DG3; antibody clone: REA897; antibody clone: REA820; antibody clone: REA753; antibody clone: REA816; antibody clone: 293C3; antibody clone: AC141 ; antibody clone: AC133; antibody clone: 7; aptamer A15; aptamer B19; HCG (Protein/Ligand); Luteinizing hormone (LH Protein/Ligand); or a binding fragment derived from any of the foregoing.

74. The AuNP of claim 70, wherein the nuclease and targeting ligand are linked through an amino acid linker.

75. The AuNP of claim 74, wherein the amino acid linker comprises a direct amino acid linker, a flexible amino acid linker, or a tag-based amino acid linker.

76. The AuNP of claim 70, wherein the nuclease and targeting ligand are linked through polyethylene glycol (PEG).

77. The AuNP of claim 70, wherein the nuclease and targeting ligand are linked through an amine- to-sulfhydryl crosslinker.

78. A composition comprising the AuNP of claim 8 and a biological sample comprising a selected cell population.

79. The composition of claim 78, wherein the biological sample comprises a selected cell population comprising a blood cell selected from 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), an endothelial cell (EC), a stromal cell, and/or a bone marrow fibroblast.

80. The composition of claim 79, wherein the blood cell comprises a CD34+CD45RA CD90+ HSC; a CD347CD133+ HSC; an LH+ HSC; a CD34+CD90+ HSPC; a CD34+CD90+ CD133+ HSPC; and/or an AHR+ HSPC.

81. The composition of claim 79, wherein the blood cell comprises a CD3+ T cell and/or a CD4+ T cell.

82. The composition of claim 78, wherein the biological sample comprises peripheral blood, bone marrow, granulocyte colony stimulating factor (GCSF) mobilized peripheral blood, and/or plerixafor mobilized peripheral blood.

83. The composition of claim 78, wherein AuNP is within the biological sample in an amount of 1 , 2, 3, 4, 5, 8, 10, 12, 15, or 20 pg of AuNP per milliliter (ml_) of biological sample.