Reduced and minimal operational manufacture of genetically modified cells

Abstract

The invention relates to reduced and minimal operational manufacture of genetically modified cells. Nanoparticles for genetically modifying selected cell types in biological samples that have been subjected to reduced or minimal manipulation are described. The nanoparticles deliver all components required for precise genomic engineering and overcome many disadvantages associated with current clinical practices to genetically engineer cells for therapeutic purposes.

Description

[0001] This application is a divisional application of the invention patent application with the application date of December 5, 2019, application number "201980088336.9", and name "Reduction and Minimum Operation Manufacturing of Genetically Modified Cells".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 775,721, filed on December 5, 2018, which is incorporated herein by reference in its entirety as if fully set forth herein.

[0004] Statement regarding sequence listing

[0005] The Sequence Listing associated with this application is provided in XML format. Technical Field

[0006] The present disclosure provides nanoparticles for genetically modifying selected cell types with reduced or minimal manipulation. The nanoparticles deliver all components required for precise genome engineering and overcome many of the shortcomings associated with current clinical practice to genetically engineer cells for therapeutic purposes. Background Art

[0007] Patient-specific gene therapy has great potential for treating genetic, infectious, and malignant diseases. For example, retroviral-mediated gene addition to hematopoietic stem cells (HSC) and hematopoietic stem and progenitor cells (HSPC) has demonstrated therapeutic results for several genetic diseases in the past 10 years, including inherited immunodeficiencies (e.g., X-linked and adenosine deaminase-deficient severe combined immunodeficiency (SCID)), hemoglobinopathies, Wiskott-Aldrich syndrome, and metachromatic leukodystrophy. In addition, this treatment approach has also improved the results of poor prognosis diagnoses such as glioblastoma. In contrast to cells from donors, the use of gene-corrected autologous or "self" cells eliminates the risk of graft-host immune response, thereby eliminating the need for immunosuppressive drugs.

[0008] The systems currently used in clinical medicine lack optimal methods for delivering gene editing components to HSCs and HSPCs and other blood cell types. For example, the CRISPR-Cas9 platform is one method used for gene editing in HSPCs in clinical settings. If the goal is gene disruption, only electroporation is required to deliver the gene editing components. However, electroporation is toxic to many cell types, and this toxicity is particularly problematic for therapies using HSCs and / or HSPCs when the starting cell number is low.

[0009] If the purpose is to insert new genetic material, a DNA template for homology-directed repair must be included. If the new genetic material is small, this can be achieved by electroporation in a single-stranded DNA (ssDNA) template, but for larger templates, the use of adeno-associated viral vectors (AAV) is currently the gold standard in clinical practice. Whether electroporation is used alone or in combination with AAV, there is no guarantee that all individual gene editing components to be delivered will be delivered to the same cell. In addition, electroporation relies on mechanical destruction and permeabilization of the cell membrane, thus compromising the viability of the cells, making them less than ideal for therapeutic use. In addition, like virus-based methods, electroporation cannot selectively deliver genes from a heterogeneous pool to a specific cell type, so a cell selection and purification process must be performed before it. The cell selection and purification process is a harsh process that leads to undesirable high levels of toxicity. Finally, AAV treatment has the potential for immunogenicity when cells are reperfused.

[0010] Any improved method of delivering gene editing components that can simplify the steps required and ensure delivery of all components to the intended cell type would be a significant improvement in clinical medicine. Nanoparticles such as polyplexes and lipoplexes have been proposed, but these have been shown to be toxic, demonstrate limited gene editing component delivery efficiency, and limited gene editing efficacy in HSCs and HSPCs. Summary of the invention

[0011] The present disclosure provides nanoparticles (NP) that allow selective genetic modification of selected cell types with reduced and minimal operations. Reduced operations mean that electroporation and viral vectors, such as AAV, are not required. Minimal operations mean that electroporation, viral vectors, and cell selection and purification processes are not required. In addition, the present disclosure also provides NPs that are specifically engineered to deliver all components required for genome editing. NPs can be used for therapies that require loss-of-function mutations, but importantly, all components required for gene addition or correction of specific mutations can also be provided. The described method is safe (i.e., no off-target toxicity), reliable, scalable, easy to manufacture, synthetic, and plug-and-play (i.e., the same basic platform can be used to deliver different therapeutic nucleic acids). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Many of the drawings filed herein are better understood in color. Applicants consider color versions of the drawings to be a part of their original filing and reserve the right to present color images of the drawings in future proceedings.

[0013] Figures 1A-1C .( Figure 1A )Current systems for ex vivo gene editing in clinical use lack optimal delivery methods for HSCs, HSPCs, and other blood cells. Figure 1A ), the current clinically used protocol includes 8 steps: (1) mobilization and apheresis; (2) immunomagnetic separation of target cell types (e.g., Figure 1A (3) stimulating the isolated cells in culture medium with recombinant growth factor (rhGF); (4) electroporating the cells to deliver gene editing components (e.g., Figure 1A (5) incubating cells in culture medium and rhGF after electroporation; (6) using a viral vector carrying a gene editing donor template (e.g., Figure 1A (7) further incubation of cells in culture medium and rhGF; and (8) harvesting of cells for reinfusion into the conditioned patient. The goal of clinical medicine is to reduce and minimize the manufacturing process. Figure 1B ) Reduced-operation manufacturing does not require electroporation or viral vector delivery, but can still utilize target cell purification processes. Figure 1B ) as shown, the NPs disclosed herein can be used to reduce the Figure 1A )'s steps 3-6. Figure 1C ) In some embodiments, minimal manipulation ex vivo manufacturing does not require isolation of selected cell types, electroporation, or viral-mediated delivery of gene editing components, thus greatly improving the efficiency of ex vivo cell manufacturing. The NPs disclosed herein with targeting ligands further reduce Figure 1A Steps 2-7 are dependent on, and do not require the use of, cell selection and purification procedures.

[0014] Figure 2 (Prior art).CD34+CD45RA-CD90+ cells are responsible for blood repopulation. Non-human primate CD34+ cells were separated into fractions i (CD45RA-CD90+), ii (CD45RA-CD90-) and iii (CD45RA+CD90-) by flow sorting, then transduced with LV encoding green fluorescent protein, mCherry or mCerulean and transplanted into bone marrow ablated autologous recipients. In all cases, blood cell engraftment corresponded only to CD34+CD45RA-CD90+ (fraction i) cells.

[0015] Figure 3 (Prior Art). Logarithmic correlation of transplanted CD34 high CD45RA-CD90+ cells / kg body weight with neutrophil and platelet engraftment (Spearman rank correlation coefficient R2: 0.0-0.19 = very weak, 0.20-0.39 = weak, 0.4-0.59 = moderate, 0.6-0.79 = strong, 0.8-1.0 = very strong). Linear regression and 95% confidence intervals are represented by solid and dashed lines, respectively.

[0016] Figure 4 When administered to humans, AuNP size determines the target tissue / elimination pathway.

[0017] Figures 5A-5D .Schematic diagram showing the synthesis and structure of NPs. ( Figure 5A ) Schematic representation of an early production scheme for gold nanoparticles (AuNPs), a scalable synthetic delivery scaffold with established in vivo compatibility. ( Figure 5B ) Schematic diagram of a synthetic method for producing and loading AuNPs with exemplary gene editing components. One depicted AuNP shows crRNA attached to the surface of the AuNP. Cpf1 nuclease and ssDNA are then attached to the crRNA. Another depicted AuNP shows crRNA attached to an 18-ethylene glycol spacer with a thiol modification attached to the surface of the 19nm AuNP core. CRISPR nuclease is attached to the cRNA to form RNPs. The AuNPs are coated with low molecular weight (MW (e.g., 2000)) polyethyleneimine (PEI). ssDNA is laminated onto the PEI-coated surface. ( Figure 5C )Schematic diagram of the Au / CRISPR NP assembly process. 1) AuNP core is synthesized and purified. 2) crRNA with spacer arm and thiol group is conjugated to the surface of gold (Au) core. 3) RNP complex is formed on the surface through the interaction of CRISPR nuclease with crRNA. 4) RNP complex is coated with 2K MW PEI. 5) ssDNA template is captured on the surface through electrostatic interaction with PEI. ( Figure 5D ) depicts another schematic diagram of the AuNPs described herein.

[0018] Figures 6A-6E .Exemplary AuNPs with selected cell targeting ligands. ( Fig. 6A ) Depiction of an exemplary AuNP configured with all components for gene addition and cell targeting. The components described include crRNA, Cpf1 nuclease, and single-stranded DNA (ssDNA) to provide a therapeutic nucleic acid sequence (e.g., a gene or a corrective portion thereof). The targeting ligand includes an aptamer. ( Figure 6B ) Schematic diagram of an alternatively formulated "layered" AuNP that can be used to deliver large oligonucleotides, such as a donor template including a homology directed repair template (HDT), a therapeutic DNA sequence, and other potential elements. The donor template is further from the AuNP surface than the depicted ribonucleoprotein complex (RNP). An aptamer targeting ligand is also depicted. ( Figure 6C ) Figure 5D The design shown in , where the aptamer targeting ligand is linked to the nuclease via a direct amino acid linker. ( Fig.6D ) Figure 5D The design shown in , where the aptamer targeting ligand is linked to the nuclease via a polyethylene glycol (PEG) tether. ( Fig. 6E ) Figure 5D The design shown in , wherein the antibody targeting ligand is linked to the nuclease via an amine-thiol crosslinker or a direct amino acid linker. Antibody targeting ligands linked via a PEG tether are also provided.

[0019] Fig. 7A , 7B .Targeted locus on the CCR5 gene. ( Fig. 7A ) The target locus has PAM sites for both Cpf1 and Cas9, with a 20 bp guide segment in the middle (SEQ ID NO: 1). Figure 7B ) HDT (SEQ ID NO: 2) was designed around the cleavage site using an 8 bp Not1 recognition sequence insert and 40 bp long symmetric homology arms.

[0020] Fig. 8A , 8B .Targeted locus within the γ-globin gene promoter. ( Fig. 8A ) The target locus has PAM sites for both Cpf1 and Cas9, with a 21 bp guide segment in the middle (SEQ ID NO: 3). Figure 8B ) HDT (SEQ ID NO: 4) was designed around the cleavage site with a 13 bp HPFH deletion and 30 bp long symmetric homology arms.

[0021] Fig. 9 The fully loaded AuNPs were monodisperse and showed good zeta potential.

[0022] Figures 10A-10D . Graphs and digital images showing characteristic properties and optimal loading concentration of synthesized AuNPs. ( Fig. 10A ) Localized surface plasmon resonance (LSPR) peak of synthesized AuNPs. ( Fig. 10B )LSPR peaks of AuNP and Au / CRISPR NP. ( Fig. 10C ) Gel electrophoresis showing the optimal AuNP / ssDNA w / w loading ratio. ( Fig. 10D )The loading concentration of Au / CRISPR NPs.

[0023] Fig.11A , 11B. Optimal loading concentration. ( Fig.11A )AuNP / crRNA 50nm (ratio 6); AuNP / crRNA 15nm (ratio 1); and AuNP / crRNA / Cpf1 / PEI / DNA 15nm (ratio 0.5). ( Fig. 11B) With the same starting reagent amount, smaller AuNPs increase the available surface area three-fold. By reducing the size, the surface area and conjugation ratio of the NPs increase.

[0024] Figures 12A-12E .(12A) CRISPR components were conjugated to AuNPs layer by layer. Fig. 12B ) Dynamic light scattering characterization of AuNPs after each layering step. Sharp single peaks and size changes after adding each layer indicate precise attachment to the surface. ( Fig. 12C ) Average size (Z-average, histogram plotted on the right axis) and polydispersity index (PDI, points plotted on the left axis) of AuNPs after each delamination step. PDI values ​​< 0.2 indicate high monodispersity without aggregation. Data are mean ± s.e. (n = 3). Fig.12D )The red shift in the LSPR of AuNPs after adding each component confirms the material loading. Fig.12E ) Zeta potential measurements after adding each layer changed from -26 mV for AuNPs to +27 mV for the final Au / CRISPR NPs. Data are mean ± s.e. (n = 3).

[0025] Figures 13A-13D Characterization of the optimal amount of Cpf1 and ssDNA. Fig.13A ) Size analysis of NPs at different AuNP / Cpf1 w / w ratios. Measurements were performed in triplicate. Fig. 13B ) Z-average values ​​and PDI values ​​for different AuNP / Cpf1 w / w ratios. It was found that the AuNP / Cpf1 w / w ratio of 0.6 was optimal in terms of size and PDI. The measurements were performed in triplicate. ( Fig. 13C ) Size analysis of NPs at different AuNP / ssDNA w / w ratios. Measurements were performed in triplicate. Fig.13D ) Z-average values ​​and PDI values ​​for different AuNP / ssDNA w / w ratios. An AuNP / ssDNA w / w ratio of 1 was found to be optimal in terms of size and PDI. The measurements were performed in triplicate.

[0026] Figures 14A-14E .Au / CRISPR NP can deliver CRISPR components to the nucleus of HSPCs. ( Fig.14A )HSPCs absorbed fully loaded AuNPs in vitro. Fig. 14B ) Nuclei of primary human CD34+ HSPCs after addition of Au / CRISPR NPs to the culture (blue, Hoechst). Fig. 14C ) Fluorophore-labeled crRNA (green, Alexa488) was used to track cellular biodistribution in the cytoplasm and nucleus. Fig.14D) Fluorophore-labeled ssDNA (red, Alexa660) is also present in the cytoplasm and nucleus. The small vesicles visible on the far left of the image indicate passive uptake via endocytosis. ( Fig.14E ) Overlay of all three stainings shows co-localization of crRNA and ssDNA. Images were obtained by confocal microscopy in Z-stack mode and 60x magnification.

[0027] Figures 15A-15C .Au / CRISPR NPs are non-toxic to primary human CD34+ HSPCs. ( Fig.15A , 15B ) Live-dead viability assay results after 24 hours (upper panel) and 48 hours (lower panel). The cell viability of the Au / CRISPR NP-treated group was higher than 70% and was similar to that of the mock-treated group. Fig. 15C ) Cell viability measured by trypan blue dye exclusion. The results of this assay correlated closely with those of the live-dead assay.

[0028] Figures 16A-16D .The figure shows the gene cutting efficiency in K562 cells and CD34+ cells. ( Fig.16A ) Viability percentage after delivery using AuNP and electroporation methods. ( Fig. 16B ) Administration dosage of CRISPR components. Fig. 16C , 16D ) Results of the Tracking Indels by Decomposition (TIDE) assay show the percentage of cleavage efficiency in K562 cells and CD34+ cells.

[0029] Fig.17 , up to 10% gene editing and HDR were observed in vitro in primary CD34+ cells obtained from healthy adult donors mobilized by G-CSF. CD34+ cells were thawed using a rapid thawing method and cultured overnight in Iscove's modified Dulbecco's medium (IMDM) containing 10% FBS and 1% Pen / Strep. The next morning, AuNPs were seeded and assembled as follows: seeding; crRNA with a PEG spacer was added to prevent electrostatic repulsion; Cpf1 protein was added and RNPs were allowed to form; coated with 2K branched PEI and single-stranded oligonucleotides (ssODN). In this example, except for the addition of terminal thiols, there was no chemical modification of crRNA to promote covalent bonding to the AuNP surface for connection. ssODN was used as HDT, here an 8bp insert using a Notl site, flanked by 40nt homologous (symmetrical) to the CCR5 target locus. The formulated AuNPs were added to the cells and incubated for 48 hours under gentle plate mixing. After 48 hours, cells were harvested, washed, and genomic DNA (gDNA) was isolated for PCR amplification and analysis.

[0030] Fig.18 .TIDE assay results show indels after editing with Au / CRISPR NPs (15nm, 50nm and 100nm) in CD34+ cells.

[0031] Figures 19A-19C . In vitro analysis of cells transplanted into NSG mice. Fig.19A ) At transplantation, 10% HDR was observed by TIDE with no significant indels at the target locus in human CD34+ cells. Fig.19B ) Restriction digestion by both T7 endonuclease I (T7EI) and NotI was observed only in cells receiving fully loaded AuNPs. ( Fig.19C ) Interestingly, an increase in colony forming ability was noted for this donor only when cells were treated with AuNPs. No significant differences were observed in the types of colonies formed under each condition.

[0032] Fig. 20 .Early post-transplantation analysis indicated engraftment of gene-edited cells. Peripheral blood was collected 6 weeks after transplantation for gDNA analysis. Of all mice treated with fully loaded AuNPs, 7 / 10 showed 0.5-6% detectable editing by TIDE. In one mouse (5% total editing), 1.7% HDR was observed by TIDE analysis.

[0033] Figures 21A-21D Optimization of HDR conditions and optimal editing dose. Fig.21A ) HDT designed for non-target strands showed higher levels of NotI insertion. Data are mean ± SE (n = 3). Fig. 21B ) shows T7EI and NotI restriction enzyme digestion of the relevant digestion bands. ( Fig. 21C ) Effects of different Au / CRISPR NP concentrations on HDR in primary human HSPCs. Data are mean ± SE (n = 3). Fig.21D ) Concentrations exceeding 20 μg / mL were toxic to CD34+ cells. Data are mean ± se (n=3). Statistical significance was determined by two-sample t-test.

[0034] Figures 22A-22C .Effects of different serum conditions and transfection components on gene editing. ( Fig.22A ) Cell viability after 48 h of treatment under different conditions. Data are mean ± SE (n = 3). Fig. 22B ) Total editing levels measured by TIDE. Data are mean ± SE (n = 3). Fig. 22C ) HDR levels measured by TIDE. Data are mean±s.e. (n=3).

[0035] Figures 23A-23F .Au / CRISPR NP carrying Cpf1 outperforms Cas9 in HDR. ( Fig.23A ) Total editing results determined by TIDE. Au / CRISPR NPs increased the cleavage efficiency of Cas9 at the CCR5 locus. Data are mean ± SE (n = 3). Fig. 23B ) HDR results determined by TIDE showed higher levels of NotI insertion using Cpf1 compared to Cas9. HDR levels observed for both Cpf1 and Cas9 delivered by Au / CRISPR NPs were higher than electroporation. Data are mean ± s.e. (n = 3). Statistical significance was determined by two-sample t-test. ( Fig.23C )Miseq analysis confirmed the trends observed by TIDE assay. Data are mean ± SE (n = 3). Statistical significance was determined by two-sample t-test. Fig.23D ) Cell viability of CD34+ cells after treatment with CRISPR Cpf1 and Cas9 using Au / CRISPR NP and electroporation methods. Cell viability was higher than 70% in all study groups. Data are mean ± SE (n = 3). Statistical significance was determined by performing one-way ANOVA. ( Fig.23E ) shows the results of colony forming cell (CFC) assay of total colony number. Data are mean±s.e. (n=3). Fig.23F ) CFC assay results show the percentage of different colonies. Data are mean ± SE (n = 3).

[0036] Fig.24A , 24B . Re-plating CFC assay shows the effect of treatment on the colony forming potential of long-term progenitor cells. ( Fig.24A ) shows the CFC assay results of total colony numbers. Data are mean ± SE (n = 3). Fig. 24B ) shows the results of CFC assay of the percentage of different colonies. Data are mean±s.e. (n=3).

[0037] Fig.25 . HDR results of the targeted locus within the γ-globin gene promoter analyzed by Miseq showed that the 13 bp deletion profile of Cpf1 was higher than that of Cas9. Data are mean±s.e. (n=3).

[0038] Fig.26 AuNP treated CD34+ cells were implanted in vivo. Fig.17The same procedure was described, except that the CD34+ cells were initially obtained from a different human donor. After 48 hours, the cells were harvested, washed, and injected into sublethally irradiated adult (8-12 weeks) NSG mice. Cell stocks were used to evaluate plate colony assays and gDNA was isolated for PCR amplification and analysis.

[0039] Figures 27A-27G .AuNP treatment enhances HSPC engraftment in NSG mice. ( Fig.27A , 27B ) Engraftment as measured by the percentage of cells expressing human CD45 in the peripheral blood of NSG recipients. AuNP- and Au / CRISPR-HDT-NP-treated cells engrafted better than mock-treated cells. Data are mean ± SE (n = 10 for Au / CRISPR-HDT-NP, n = 10 for AuNP, n = 5 for mock, n = 4 for non-injected). Statistical significance was determined by two-sample t-test. ( Fig.27C ) Human CD20+ B cell engraftment kinetics in peripheral blood. Fig.27D ) Human CD14+ monocyte engraftment kinetics in peripheral blood. Fig.27E ) Human CD3+ T cell engraftment kinetics in peripheral blood. Fig.27F ) shows the CFC assay of total colony counts of bone marrow samples. CFC results correlated well with engraftment results. Data are mean ± SE (n = 3). Statistical significance was determined by two-sample t-test. ( Figure 27G ) shows the CFC measurement results of the frequencies of different morphologies. Data are mean±se (n=3).

[0040] Fig.28 The weight of mice was stable during the study. The weight of mice in different cohorts was tracked. Data are mean ± SE (n = 10 for Au / CRISPR-HDT-NP, n = 10 for AuNP, n = 5 for mock, n = 4 for non-injected).

[0041] Figures 29A-29D . Engraftment levels of cell populations in autopsy samples after treatment with Au / CRISPR NPs. Fig.29A ) Engraftment levels in the bone marrow. Data are mean ± SE (n = 10 for Au / C RISPR-HDT-NPs, n = 10 for AuNPs, n = 5 for mocks). Fig.29B ) Engraftment levels in spleen. Data are mean ± s.e. (n = 10 for Au / CRISPR-HDT-NPs, n = 10 for AuNPs, n = 5 for mocks). Fig.29C) Engraftment levels in the thymus. Data are mean ± SE (n = 10 for Au / CRISPR-HDT-NPs, n = 10 for AuNPs, n = 5 for mock). Fig.29D ) Engraftment levels in peripheral blood. Data are mean ± SE (n = 10 for Au / CRISPR-HDT-NPs, n = 10 for AuNPs, n = 5 for mock).

[0042] Fig. 30A , 30B .( Fig. 30A ) Colony forming potential of Au / CRISPR NP treated cells before implantation. CFC assay of total colony number before implantation is shown. Data are mean ± SE (n = 3). Statistical significance was determined by two-sample t-test. ( Fig. 30B ) shows the results of CFC assay of the percentage of different colonies. Data are mean±s.e. (n=3).

[0043] Fig.31 Representative colony morphology after treatment with Au / CRISPR NPs. Burst forming units-erythrocytes (BFU-E), granulocytes-monocytes (GM).

[0044] Figures 32A-32E .Continued editing levels after implantation. ( Fig.32A ) TIDE measurement results of total editing and HDR levels before implantation. ( Fig.32B ) Tracking of total editor levels. Peripheral blood samples were collected every other week starting from 4 weeks after transplantation. Data are mean ± SE (n = 10). Fig.32C ) Tracking of HDR levels after implantation. Data are mean ± SE (n = 10). Fig.32D ) Total editing levels in peripheral blood, bone marrow, and spleen at autopsy. Data are mean ± SE (n = 10). Fig.32E ) HDR levels in peripheral blood, bone marrow and spleen at autopsy. Data are mean±s.e. (n=10).

[0045] Fig.33 .Digestion with NotI and T7EI restriction enzymes after treatment with Au / CRISPR NPs.

[0046] Fig.34 .sequences of crRNA, HDT and primers (SEQ ID NO:5-19).

[0047] Figures 35A-35D .( Fig.35A ) Potential off-target cleavage sites of Cpf1 and Cas9 and γ-globin target site on CCR5 (SEQ ID NO: 20-27). Fig.35B) Cas9 and Cpf1 guide sequences and HDR templates for hereditary persistent fetal hemoglobin (HPFH) (SEQ ID NOs: 28-52 and 214-224). Each guide sequence spans a specific mutation. A target DNA sequence that can be used for crRNA synthesis is provided. ( Fig.35C ) RNA sequences (SEQ ID NOs: 225-262) transcribed from DNA target sites (SEQ ID NOs: 20-22, 24-26, 28-32, 42, 43, 84-97, and 214-224) for genetic engineering. Fig.35D ) Table 1 provides DNA target sites, cRNA sequences, and complementary sets of HDTs.

[0048] Fig.36 . Other sequences supporting the present disclosure (SEQ ID NO: 112-138). DETAILED DESCRIPTION

[0049] Gene therapy has great potential for treating genetic, infectious, and malignant diseases. For example, retroviral-mediated gene addition to hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs) has demonstrated therapeutic results for several genetic diseases in the past 10 years, including inherited immunodeficiencies (e.g., X-linked and adenosine deaminase-deficient severe combined immunodeficiency (SCID)), hemoglobinopathies, Wiskott-Aldrich syndrome, and metachromatic leukodystrophy. In addition, this treatment approach has improved the results of poor prognosis diagnoses such as glioblastoma. The use of gene-corrected autologous or "self" cells, rather than cells from a donor, eliminates many of the risks of cell-based genetic therapies, including graft-host immune responses, thereby eliminating the need for immunosuppressive drugs.

[0050] Currently, clinical systems lack optimal methods for delivering gene editing components to many cell types. For example, for hematopoietic stem cells (HSCs) and HSCs

[0051] Progenitor cells (HSPC), the current state-of-the-art technology includes removing cells from patients through bone marrow aspirates or mobilized peripheral blood, sorting the large population into autologous HSPCs by immune selection of cells expressing the surface marker CD34, and then culturing these cells in the presence of cytokines. If the purpose is to destroy problematic genes, electroporation is used to deliver gene editing components to cells. Electroporation generally refers to applying an electric field to cells to increase the permeability of the cell membrane, thereby allowing molecules to pass through and be introduced into the cells. Electroporation is toxic to many cell types, and in the case of low starting cell numbers, this toxicity is particularly problematic for therapies using HSC and / or HSPC.

[0052] If the purpose is to insert new genetic material into the cell, a DNA template for homology-directed repair must be included. If the new genetic material is small, this can be achieved by electroporation alone, but for larger forms of genetic material, the additional use of adeno-associated viral vectors (AAV) is currently the gold standard in clinical practice. There are still known risks of genotoxicity and other limitations associated with the use of viral vectors for gene transfer. For example, the development of malignancies caused by insertional mutagenesis in patients treated with HSPC gene therapy demonstrates the risk of genotoxicity. This adverse side effect stems from the semi-random nature of retroviral-mediated transgene delivery into the host cell genome. The dysregulation of nearby genes by the inserted transgenic sequence is the molecular basis for the clonal expansion and malignant transformation observed in some gene therapy patients, but the interaction between the inserted transgene and the surrounding genomic environment can also lead to transgene attenuation or silencing, thereby reducing the therapeutic effect. Other limitations associated with the use of specific viral vectors include the induction of immune responses, reduced efficacy over time in dividing cells (e.g., adeno-associated vectors), the inability to adequately target selected cell types in vivo (e.g., retroviral vectors), and, as indicated, the inability to control the insertion site and number of insertions (e.g., lentiviral vectors).

[0053] Recent years have seen the emergence of gene editing as a safer alternative to retroviral-mediated gene transfer, made possible by the development of engineered guide RNAs and nucleases that target specific DNA sequences and predictably generate DNA double-strand breaks (DSBs) at the targeted sequences. To date, these programmable complexes have been most effective in providing promising therapies when problematic genes need to be removed or silenced (i.e., generating loss-of-function mutations). This is because DSBs are most often repaired by error-prone non-homologous end joining (NHEJ), which results in oligonucleotide insertions and deletions (indels) at the DSB site.

[0054] For the correction of gene addition or specific mutations, the less common homology-directed repair (HDR) of DSB is required. In this case, a more complex payload including engineered guide RNA and nuclease and homology-directed repair template must be delivered together. The proof of concept of this method has been confirmed in HSPC, but it also requires tandem electroporation of some gene editing components, followed by transduction with non-integrating viral vectors, particularly recombinant adeno-associated virus (rAAV) vectors to deliver DNA templates, or simultaneous electroporation of engineered nuclease components at a specified cell concentration with chemically modified single-stranded oligonucleotide templates. In addition, each engineered guide RNA, nuclease, and homology-directed repair template must be uniquely engineered for each specific genetic target, which requires separate evaluation of delivery, activity, and specificity in cell lines and HSPCs.

[0055] Whether electroporation is used alone or in combination with AAV, there is no guarantee that all individual components required for gene editing will be delivered to the same cell. In addition, electroporation and many viral vectors cannot selectively deliver genes from a heterogeneous pool to a specific cell type, so cell selection and / or purification processes must be performed before these treatments. Cell selection and purification processes are operations that can cause cytotoxicity or loss of fitness. An example of this is blood stem cells, which can begin to differentiate when manipulated, resulting in a loss of engraftment potential because more differentiated blood cells cannot support long-term blood production.

[0056] Therefore, despite many exciting breakthroughs in the ability to target genetic therapies at specific sites within the genome, the continued lack of safe and effective delivery vehicles has hampered the clinical translation of gene editing systems, particularly for HSCs / HSPCs.

[0057] Any improved method of delivering gene editing components to cells that reduces toxicity and can simplify the steps required to ensure that all gene editing components are delivered to the cells would be a significant improvement in clinical medicine. From a logistical perspective, also given the complex infrastructure required to operate autologous cell products, having a more localized and streamlined manufacturing process would reduce vein-to-vein time, which may be important in certain disease situations. Nanoparticles such as polyplexes and lipoplexes have been proposed, but these have been shown to be too toxic to cells and have demonstrated limited efficiency in delivering gene editing components to, for example, HSPCs.

[0058] The present disclosure provides nanoparticles (NP) that allow selective genetic modification of selected cell types with reduced and minimal operations. Reduced operations mean that electroporation and viral vectors, such as AAV, are not required. In specific embodiments, reduced operations mean that electroporation and viral vectors, such as AAV, are not required. Minimal operations mean that electroporation, viral vectors, and cell selection and purification processes are not required. In specific embodiments, minimal operations mean that electroporation, viral vectors, and cell selection and purification processes are not required. In specific embodiments, minimal operations mean that before being exposed to NP disclosed herein, only samples containing selected blood cell types are washed to remove platelets. As will be described in more detail elsewhere herein, whether NP is used for reduced operations or minimal operation processes depends on whether cell targeting ligands are associated with NP.

[0059] Targeting ligands include, for example, antibodies, aptamers, ligands or other molecules that specify NPs to interact with target cell types. The selected cell targeting ligand may include surface-anchored targeting ligands that selectively bind NPs to selected cells and trigger cellular uptake. In a specific embodiment, cellular uptake may be mediated by receptor-induced endocytosis. As disclosed in more detail elsewhere herein, the selected cell targeting ligand may include antibodies, scFv proteins, DART molecules, peptides and / or aptamers. Specific embodiments utilize antibodies, antibody binding fragments or aptamers that recognize CD3, CD4, CD34, CD90, CD133, CD164, luteinizing hormone releasing hormone (LHRH) receptor, aryl hydrocarbon receptor (AHR) or CD46 to target HSC. Specific embodiments include one or more of anti-human CD3 antibody, anti-human CD4 antibody, anti-human CD34 antibody, anti-human CD90 antibody, anti-human CD133 antibody, anti-human CD164 antibody, anti-human CD133 aptamer, human luteinizing hormone, human chorionic gonadotropin (hCG, a ligand of the LHRH receptor), degarelix acetate (an antagonist of the LHRH receptor), or StemRegenin 1 (a ligand of the AHR) as a targeting ligand.

[0060] When the disclosed NPs are added to a heterogeneous mixture of cells (e.g., an ex vivo blood product), the engineered NPs bind to the selected cell population and are internalized into the target cells. This process provides an entry for the genetic engineering components carried by the NPs, so the selected cells are genetically modified. Providing all the components required for genetic engineering on a single particle ensures that the cells that ingest the particles receive all the necessary components rather than a subset thereof. By targeting the NPs to the desired cell population, cell selection (immunomagnetic or other) is no longer necessary.

[0061] The use of the NPs disclosed herein accelerates the ex vivo manufacturing of therapeutic cells and produces less cell damage during processing and genetic engineering. In certain embodiments, this method also reduces the amount of time from the harvest of patient cells to the reinfusion of genetically modified blood cell products.

[0062] In a specific embodiment, NP disclosed herein is a gold nanoparticle (AuNP). Specifically, AuNP has been shown to be non-toxic to non-dividing and dividing mammalian cells, and has been applied to the in vivo delivery of RNA therapeutics in clinical trials. In addition, due to their unique surface chemistry, AuNP can be loaded with all components required for gene editing. As described in more detail herein, gene editing components can be connected to NP in a specially designed hierarchical structure, which optimizes the function and characterization of NP in terms of, for example, size, polydispersity index, and gene editing efficiency.

[0063] Specific embodiments include NPs with components that provide targeted loss-of-function mutations. These embodiments include targeting elements (e.g., guide RNA) and cutting elements (e.g., nucleases) associated with the NP surface. In specific embodiments, the targeting element is conjugated to the surface of NP via a thiol linker. In specific embodiments, the targeting element and / or cutting element are conjugated to the surface of NP via a thiol linker. In specific embodiments, the targeting element is conjugated to the surface of NP via a thiol linker, and the cutting element is connected to the targeting element to form a ribonucleoprotein (RNP) complex. The targeting element targets the cutting element to a specific site for cutting and NHEJ repair.

[0064] Specific embodiments include NPs with components that provide targeted gain-of-function mutations (e.g., gene addition or correction). In specific embodiments, these embodiments include metal NPs (e.g., AuNPs) associated with targeting elements, cutting elements, homology-directed repair templates (HDTs), and therapeutic DNA sequences. The targeting element targets the cutting element to a specific site for cutting, and the homology-directed repair template provides HDR repair, wherein after HDR repair, the therapeutic DNA sequence has been inserted into the target site. The homology-directed repair template and the therapeutic DNA sequence together may be referred to as donor templates herein. In specific embodiments, the targeting element is conjugated to the surface of the NP via a thiol linker. In specific embodiments, the targeting element and / or the cutting element are conjugated to the surface of the NP via a thiol linker. In specific embodiments, the targeting element is conjugated to the surface of the NP via a thiol linker, and the cutting element is connected to the targeting element to form a ribonucleoprotein (RNP) complex. In these embodiments, the RNP complex is closer to the surface of the NP than the donor template material. When, for example, the targeting element and / or the cutting element are bacterial sources, this construction is beneficial. This is because many individuals who may receive NP described herein may have pre-existing immunity to components of bacterial origin (such as gene editing components of bacterial origin). Including gene editing components of bacterial origin on the inner layer of the fully formulated NP allows components of non-bacterial origin (e.g., donor templates) to shield components of bacterial origin (e.g., targeting elements and / or cutting elements) from the patient's immune system. This protects components of bacterial origin from attack, and also avoids or reduces undesirable inflammatory responses to NP after administration. In addition, this can allow repeated administration of NP in vivo without being inactivated by the host immune response.

[0065] Specific embodiments can utilize AuNPs associated with at least four layers, wherein the first layer comprises CRISPR (clustered regularly interspaced short palindromic repeats) guide RNA (crRNA), the second layer comprises a nuclease, the third layer comprises ssDNA, and the fourth layer comprises a targeting ligand, wherein the first layer is closest to the surface of the NP core, and the second layer is second closest to the surface of the NP core. The third layer is third closest to the nanoparticle core, and the fourth layer is farthest from the NP core. In specific embodiments, a layer refers to a layer associated with NPs, which comprises components for genetic modification of a selected cell population, including crRNA, nucleases, donor templates, targeting ligands, and / or components for producing a layer including a linker and a polymer (e.g., polyethylene glycol (PEG) and polyethyleneimine (PEI)).

[0066] Particular embodiments utilize CRISPR gene editing. In particular embodiments, CRISPR gene editing can be performed using CRISPR guide RNA (crRNA) and / or CRISPR nucleases (e.g., Cpf1 (also known as Cas12a) or Cas9).

[0067] Specific embodiments use characteristic structures that increase HDR efficiency and / or accuracy. For example, Cpf1 has a short single crRNA and cuts the target DNA in a staggered form with 5'2-4 nucleotide (nt) overhangs (called sticky ends). Sticky ends are conducive to HDR, Kim et al. (2016) Nat Biotechnol.34 (8): 863-8. Moreover, before performing RNP cutting of the genome to promote HDR, the donor template should be released from NP. Therefore, in specific embodiments disclosed herein, it is found that the donor template is farther from the surface of NP than the targeting element and the cutting element. The present disclosure also unexpectedly found that delivering gene editing components on AuNP increases the efficiency and / or accuracy of HDR. Therefore, specific embodiments utilize AuNP to deliver gene editing components.

[0068] The specific material used for genetic engineering is customized for individual patients based on the desired therapeutic outcome. When the targeting ligand is not included as a component of the NP, the NP provides reduced operational manufacturing, thereby eliminating the need to utilize electroporation and viral vector delivery. The inclusion of the targeting ligand allows for minimal operational manufacturing, thereby eliminating the need to perform cell selection and purification processes.

[0069] After adding NPs to the reduced or minimally manipulated blood cell product, a period of incubation is performed. Thereafter, the cell product can be optionally washed to remove excess NPs and re-administered to the patient. In a specific embodiment, the cells can be stored. Storage can include room temperature, refrigeration (2-8°C) or cryopreservation (≤20°C, including storage in liquid nitrogen or gas phase) conditions, depending on the length of time required for the patient to be prepared for re-infusion. The biological sample can be cryopreserved before and / or after exposure to the NP before re-infusion into the patient.

[0070] Various aspects of the present disclosure are now described in the following additional details and options: (I) gene editing systems and components; (II) nanoparticles and their conjugation to gene editing components; (III) gene editing efficiency; (IV) selected cells and selected cell targeting ligands; (V) source and processing of cell populations; (VI) formulation and cryopreservation of cells; (VII) nanoparticle formulation; (VIII) kits; (IX) exemplary methods of use; (X) exemplary manufacturing schemes and comparisons; (XI) assays for evaluating nanoparticle performance; (XII) exemplary embodiments; (XIII) experimental embodiments; and (XIV) concluding paragraphs.

[0071] (I) Gene editing systems and components. Within the teachings of the present disclosure, any gene editing system capable of precise sequence targeting and modification can be used. These systems generally include a targeting element for precise targeting and a cutting element for cutting the targeted genetic site. Guide RNA is an example of a targeting element, and various nucleases provide examples of cutting elements. The targeting element and the cutting element can be separate molecules or connected, for example, by nanoparticles. Alternatively, the targeting element and the cutting element can be connected together to form a dual-purpose molecule. When the therapeutic nucleic acid sequence is intended to be inserted, the system also includes an HDR template (which may include homology arms) associated with the therapeutic nucleic acid sequence. However, as further described below, different gene editing systems can use different components and structures while maintaining the ability to accurately target, cut and modify selected genomic sites.

[0072] In a specific embodiment, the CRISPR gene editing system can be used to target sites for genetic engineering. The CRISPR nuclease system is a prokaryotic immune system that confers resistance to foreign genetic elements (such as plasmids and bacteriophages) and provides a form of acquired immunity. CRISPR is a DNA locus containing short repeated base sequences. In the case of the prokaryotic immune system, each repetition is followed by a short segment of the interval DNA of the foreign genetic elements exposed by prokaryotes. This CRISPR array of repeats interspersed with spacers can be transcribed into RNA. The RNA can be processed into a mature form and associated with Cas (CRISPR-related) nuclease. The CRISPR-Cas system includes RNA and Cas nucleases with sequences that can hybridize with foreign genetic elements, which can recognize and cut these foreign genetic elements in the genome.

[0073] Instead of generating custom proteins to target specific sequences, the CRISPR-Cas system can program a single Cas enzyme to recognize a specific DNA target through a short guide RNA molecule (crRNA). The CRISPR-Cas systems of bacterial and archaeal adaptive immunity show extreme diversity in protein composition and genomic locus structure. The CRISPR-Cas system loci have more than 50 gene families and there are no strictly universal genes, which indicates the rapid evolution and extreme diversity of locus structure. At present, a multi-pronged approach has been adopted to comprehensively identify Cas genes in 395 maps of 93 Cas proteins. The classification includes a signature gene spectrum plus a signature of the locus structure. A classification of CRISPR-Cas systems is proposed, in which these systems are roughly divided into two categories, the first category has a multi-subunit effector complex, and the second category has a single subunit effector module, such as Cas9 protein. Effective gene editing in human CD34+ cells has been demonstrated using electroporation of CRISPR / Cas9 mRNA and single-stranded oligodeoxyribonucleotides (ssODN) as donor templates for HDR. De Ravin et al. Sci Transl Med. 2017; 9(372): eaah3480. Novel effector proteins associated with class 2 CRISPR-Cas systems can be developed as powerful genome engineering tools, and prediction of putative novel effector proteins and their engineering and optimization is important. In addition to class 1 and class 2 CRISPR-Cas systems, a putative class 2 type V CRISPR-Cas class, exemplified by Cpf1, has recently been identified Zetsche et al. 2015 (Cell 163) 3(: 759-771).

[0074] Additional information about CRISPR-Cas systems and their components is described in US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, U S8889418, US8895308, US8906616, US8932814, US8945839, US 8993233, and US8999641 and related applications; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635, WO2014 / 093655, W O2014 / 093661, WO2014 / 093694, W02014 / 093701, W02014 / 093709, WO2014 / 093712, WO2014 / 093718, WO2014 / 14559 9. WO2014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726, WO2014 / 204727, WO2014 / 204728, WO2014 / 204 729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089354, WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, WO2016205711, WO2017 / 106657, WO2017 / 127807 and related applications.

[0075] The Cpf1 nuclease in particular can provide additional flexibility in target site selection through a short three-base pair recognition sequence (TTN), known as a protospacer adjacent motif or PAM. The Cpfts cleavage site is at least 18 bp away from the PAM sequence, so the enzyme can repeatedly cleave a specific locus after indels (insertions and deletions) have formed, potentially increasing the efficiency of HDR. Successful HDR results in mutation of the PAM sequence so that no further cleavage can occur. In addition, staggered DSBs with sticky ends allow for directional specific donor template insertion, which is advantageous in non-dividing cells.

[0076] As previously mentioned, specific embodiments adopt characteristic structures that increase HDR efficiency and / or accuracy. For example, Cpf1 has a short single crRNA and cuts the target DNA in a staggered form with 5'2-4 nucleotide (nt) overhangs (called sticky ends). Sticky ends are conducive to HDR, Kim et al. (2016) Nat Biotechnol.34 (8): 863-8. Moreover, before performing RNP cutting of the genome to promote HDR, the donor template should be released from NP. Therefore, in specific embodiments disclosed herein, it is found that the donor template is farther from the surface of NP than the targeting element and the cutting element. The present disclosure also unexpectedly found that delivering gene editing components on AuNP increases the efficiency and / or accuracy of HDR. Therefore, specific embodiments utilize AuNP to deliver gene editing components.

[0077] Specific embodiments may utilize engineered variant Cpf1. For example, US 2018 / 0030425 describes engineered Cpf1 nucleases with altered and improved target specificity from Lachnospiraceae bacteria ND2006 and Acidaminococcus sp. BV3L6. Specific variants include Lachnospiraceae bacteria ND2006 with mutations (i.e., replacing the natural amino acid with a different amino acid such as alanine, glycine, or serine) at one or more of the following positions: S203, N274, N278, K290, K367, K532, K609, K915, Q962, K963, K966, K1002, and / or S1003. Specific Cpf1 variants can also include Acidaminococcus BV3L6 Cpf1 (AsCpf1) with a mutation (i.e., a different amino acid, such as alanine, glycine, or serine, replacing a native amino acid (unless the native amino acid is serine)) at one or more of the following positions: N178, S186, N278, N282, R301, T315, S376, N515, K523, K524, K603, K965, Q1013, Q1014, and / or K1054. In specific embodiments, engineered Cpf1 variants include eCfpl. Other Cpf1 variants are described in US2016 / 0208243 and WO / 2017 / 184768.

[0078] Specific embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a class of site-specific nucleases engineered to bind and cleave DNA at specific locations. ZFNs are used to introduce double-strand breaks (DSBs) into specific sites in a DNA sequence, which enables ZFNs to target unique sequences within the genome in a variety of different cells. In addition, after a double-strand break, HDR or NHEJ occurs to repair DSBs, thereby achieving genome editing.

[0079] ZFNs are synthesized by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The DNA binding domain contains three to six zinc finger proteins that act as transcription factors. The DNA cleavage domain includes, for example, the catalytic domain of the Fok1 endonuclease. The Fok1 domain acts as a dimer, which requires two constructs with unique DNA binding domains for sites on the target sequence. The Fok1 cleavage domain cleaves within a five or six base pair spacer sequence that separates the two inverted half sites.

[0080] For additional information on ZFNs, see Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Bibikova, et al. Science 300, 764 (2003); Bibikova, et al. Genetics 161, 1169-1175 (2002); Miller, et al. The EMBO journal 4, 1609-1614 (1985); and Miller, et al. Nature biotechnology 25, 778-785 (2007)].

[0081] Specific embodiments can use transcription activator-like effector nuclease (TALEN) as a gene editor. TALEN refers to a fusion protein comprising a transcription activator-like effector (TALE) DNA binding protein and a DNA cleavage domain. TALEN is used to edit genes and genomes by inducing DSB in DNA, which induces repair mechanisms in cells. Typically, two TALENs must bind and flank each side of the target DNA site to dimerize the DNA cleavage domain and induce DSB. If an exogenous double-stranded donor DNA fragment is present, DSB is repaired in the cell by NHEJ or HDR.

[0082] As indicated, TALEN has been engineered to bind to a target sequence, such as an endogenous genome, and cut DNA at the location of the target sequence. TALEN's TALE is a DNA binding protein secreted by Xanthomonas bacteria. The DNA binding domain of TALE contains a highly conserved 33 or 34 amino acid repeat sequence with different residues at the 12th and 13th positions of each repeat sequence. These two positions, called repeat variable di-residues (RVDs), show a strong correlation with specific nucleotide recognition. Therefore, by changing the amino acids in the RVD and incorporating unconventional RVD amino acids, targeting specificity can be improved.

[0083] Examples of DNA cleavage domains that can be used for TALEN fusions are wild-type and variant Fok1 endonucleases. For additional information on TALEN, see Boch, et al. Science 326, 1509-1512 (2009); Moscou, & Bogdanove, Science 326, 1501 (2009); Christian, et al. Genetics 186, 757-761 (2010); and Miller, et al. Nature biotechnology 29, 143-148 (2011).

[0084] Specific embodiments utilize MegaTAL as a gene editing agent. MegaTAL has a single-chain rare-cleavage nuclease structure in which the TALE is fused to the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease functions in the same domain. In contrast to TALENs, megaTALs only require the delivery of a single peptide chain for functional activity.

[0085] Exemplary crRNAs for related genetic engineering targets include: UAAUUUCUACUCUUGUAGAUUUCGGACCCGUGCUACAACUU (SEQ ID NO:80, chr11-gsh-gRNA1); UAAUUUCUACUCUUGUAGAUAUAGAAU AGCCUCAUAUUUUA (SEQ ID NO:81, chr11-gsh-gRNA 2); UAAUUUCUACUCUUGUAGAUGAGCUGUUGGCAUCAUGUUCCUG (SEQ ID NO:82, chr11-gsh-gRNA 3); UAAUUUCUACUCUUGU AGAUUCCAAACCUCCUAAAUGAUAC (SEQ ID NO:83, chr11-g sh-gRNA 4); and UAAUUUCUACUCUUGUAGAUCACCCGAUCC ACUGGGGAGCA (SEQ ID NO:5, chr11-gsh-gRNA 5).Relevant target sites for genetic engineering include (PAM sites are indicated in italics): TTTGTGTCCCCG TTTTGGTTGGTAAAC (SEQ ID NO: 84, chr11-gsh-target 1); TTLA AAAATCAATACCGATAATAATGA (SEQ ID NO: 85, chr11-gsh-target 2); TTTCTTAATATGAATATTAATATCGGT (SEQ ID NO: 86, chr11-gsh-target 3); TTTCCGTATCTGGAAGGGGCATCTTGG (SEQ ID NO: 87, chr11-gsh-target 4); TTTCCTTAGGACCGGAAGGA TTACAGC (SEQ ID NO: 88, chr11-gsh-target 5); TTTGCCTAAAAGGCACTATGTCAAATG (SEQ ID NO: 89, chr11-gsh-target 6); TTTCCTTAAGGACCGGAAGGA TTACAGC (SEQ ID NO: 81, chr11-gsh-target 7); TGGAGCTGTTGGCATCATGTTCCTG (SEQ ID NO:90, chr11-gsh-target 7); TTTGATTCTTTTCTATCTCAGGACAGA (SEQ ID NO:91, chr11-gsh-target 8); TTLATAGACATCCCACACTGTAGTTCT (SEQ ID NO:92, chr11-gsh-target 9); TTLATTAATTTGAGAACCAAC ATAAGG (SEQ ID NO:93, chr11-gsh-target 10); TTLATTTTCTTTTTGGTAAGAAGGAAC (SEQ ID NO:94, chr11-gsh-target 11); TT TCACACACACACACACACACACACACA (SEQ ID NO:95, chr11-gsh-target 12); TT7ATCCAAACCTCCTAAATGATAC (SEQ ID NO:96, chr11-gsh-target 13); NO:96, chrl1-gsh-target 13); TT7ACACCCGATCCACTGGGGAGCA (SEQ ID NO:21, chrl1-gsh-target 14); and TT7TTGATTCTTTTCTAT CTCAGGACA (SEQ ID NO:97, chrl1-gsh-target 15). These target sites reflect genomic safe harbors (GSH) within HSPCs. In specific embodiments, these GSH sites are SEQ ID NOs:21 and 84-97 (chrl1-gsh-target 1-15) reflected above, but with 1, 2, 3 or 4 nucleotide substitutions to account for typical genetic variation in the population.

[0086] The present disclosure also provides target sites and targeting sequences that can be used to treat other conditions such as hemoglobinopathies and human immunodeficiency virus (HIV) loci (see, e.g. Fig. 7A , 7B , 8A, 8B, 34 and 35A-35D).

[0087] In a specific embodiment, NP can deliver factors that promote the desired target DNA repair pathway. The first step in any approach to repair double-stranded DNA breaks is to stabilize the free ends of DNA at the break site. DNA stabilizing proteins specific to the target repair pathway can be incorporated to promote the specific DNA repair pathway. For NHEJ, two proteins are involved in stabilizing the free ends of DNA: Ku70 and Ku80. For HDR, a three-protein complex called MRN composed of MRE11, Nbs1 and RAD50 is required. These molecules may include oligomers (mRNA) or proteins of any factors involved to ensure that cells receiving gene editing mechanisms also have these factors. Alternatively or in combination, small interfering RNA (siRNA, short hairpin RNA or microRNA) that will reduce the expression of the NHEJ pathway may also be included.

[0088] The template for HDR can be symmetrical or asymmetrical homology arms as described in Richardson et al., Nat Biotechnol. 2016; 34(3): 339-44. Each donor template can contain homology arms (HDR template) flanked by 20 bp random DNA barcode elements for clone tracking, located upstream of the human phosphoglycerate kinase (PGK) promoter that drives expression of therapeutic DNA sequences in clinical applications. Humanized Cpf1 protein can be synthesized by a commercial manufacturer (Aldevron), and guide RNAs with two modifications, an atom oligoethylene glycol spacer and a 3' terminal thiol can also be obtained from a commercial source (Integrated DNA Technologies, Coralville, IA). Single-stranded homology template DNA (ssODN) can also be synthesized by a commercial manufacturer (Integrated DNA Technologies, Coralville, IA). For examples of such sequences, see Fig. 7A , 7B , 8A, 8B, 34, 35B and 35D.

[0089] As noted, in certain embodiments, a gene editing system providing genetic therapy will comprise a guide RNA and a nuclease. In certain embodiments, a donor template may be used, particularly when performing gain-of-function therapy or precise loss-of-function therapy. In certain embodiments, a gene editing system comprises an HDR template and a therapeutic nucleic acid sequence.

[0090] All nucleic acid-based components of the gene editing system can be single-stranded, double-stranded, or can have a mixture of single-stranded and double-stranded regions. For example, the guide RNA or donor template can be single-stranded DNA, single-stranded RNA, double-stranded DNA or double-stranded RNA. In a specific embodiment using NPs described herein, the nucleic acid end farthest from the NP surface can be protected by methods known to those skilled in the art (for example, from exonucleolytic degradation). For example, one or more dideoxynucleotide residues can be added to the 3' end of the linear molecule and / or a self-complementary oligonucleotide can be connected to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad Sci USA 84: 4959-4963; Nehls et al. (1996) Science 272: 886-889. Other methods for protecting exogenous polynucleotides from degradation include adding terminal amino groups and using modified internucleotide bonds, such as phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. Chemically modified mRNA can be used to increase intracellular stability, while asymmetric homology arms and phosphorothioate modifications can be incorporated into ssODN to improve HDR efficiency. In specific embodiments utilizing NPs described herein, nucleic acids can be protected from electrostatic (charge-based) repulsion by, for example, adding charge shielding spacers. In specific embodiments, the charge shielding spacer can include an 18-atom oligoethylene glycol (OEG) spacer added to one or both ends. In specific embodiments, the charge shielding spacer can include a 10-26-atom oligoethylene glycol (OEG) spacer added to one or both ends.

[0091] The donor polynucleotide can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.

[0092] In a specific embodiment, an HDR template (HDT) is designed to be used as a template in homologous recombination, such as within or near a target sequence that is cut or cleaved by an enzyme (e.g., a nuclease) of a gene editing system. The HDR template polynucleotide may have any suitable length, such as 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 2000, 3000, 4000, 5000 or more nucleotides. In a specific embodiment, the HDR template polynucleotide is complementary to a portion of a polynucleotide comprising a target sequence. When optimally aligned, the HDR template polynucleotide overlaps with one or more nucleotides (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides) of the target sequence.

[0093] In a specific embodiment, the HDR template can include enough homology to the genomic sequence of the cleavage site, for example, 70%, 80%, 85%, 90%, 95% or 100% homology to the nucleotide sequence of the cleavage site, for example, within 50 bases or less of the cleavage site, for example, within 30 bases of the cleavage site, within 15 bases, within 10 bases, within 5 bases or directly flanking the cleavage site to support HDR between the HDR template and the genomic sequence with homology thereto. 25, 50, 100 or 200 nucleotides or more than 200 nucleotides (or any integer value or more between 10 and 200 nucleotides) with sequence homology between the HDR template and the targeted genomic sequence can support HDR. The homology arms or flanking sequences are usually identical to the genomic sequence, for example, identical to the genomic region where a double-strand break (DSB) occurs. However, absolute identity is not required.

[0094] In certain embodiments, the donor template comprises a heterologous therapeutic nucleic acid sequence flanked by two homology regions, such that HDR between the target DNA region and the two flanking sequences results in insertion of the heterologous therapeutic nucleic acid sequence into the target region. In some examples, the homology arms or flanking sequences of the HDR template are asymmetric.

[0095] As noted, in specific embodiments, the donor template comprises a therapeutic nucleic acid sequence. The therapeutic nucleic acid sequence can include a corrected gene sequence; a complete gene sequence and / or one or more regulatory elements associated with gene expression. The corrected gene sequence can be a portion of a gene that needs to be corrected, or a complete replacement copy of a gene can be provided. The corrected gene sequence can provide a complete copy of a gene without having to replace an existing defective gene. One of ordinary skill in the art will recognize that when providing a corrected copy, it may or may not be necessary to remove the defective gene. When a gene is inserted into a genetic safe harbor, the therapeutic nucleic acid sequence should include the coding region and all regulatory elements required for its expression.

[0096] Examples of therapeutic genes and gene products include skeletal protein 4.1, glycophorin, p55, Duffy alleles, 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; ubiquitin 2; C9ORF72, α2β1; αvβ3; αvβ5; αvβ63 ;BOB / GPR15;Bonzo / STRL-33 / TYMSTR;CCR2;CCR3;CCR5;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, ApoAIV, ApoE, ATM, BAI-1, BDNF, Beta*(BLU), bFGF, B LC1, 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, FUS1, 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 alpha, interferon beta, interferon gamma, 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, 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. ,

[0097] In certain embodiments, the therapeutic gene comprises a coding sequence for a therapeutic expression product (eg, protein, RNA) and all associated regulatory elements (eg, promoter, etc.) that lead to expression of the gene product.

[0098] In certain embodiments, therapeutic genetic engineering disrupts genetic loci to prevent binding. See, e.g. Fig. 8A , 8B. In a specific embodiment, the genetic engineering is based on gene editing components, including Cpf1 and a guide RNA, wherein the guide RNA targets a single nucleotide polymorphism (SNP) or a 13-nucleotide deletion overlapping the BCL11a binding site in the gamma globin locus on chromosome 11, or a SNP within an erythroid-specific enhancer element in the second intron of the BCL11a gene on chromosome 2. In a specific embodiment, the genetic engineering is based on gene editing components, including Cpf1 and a guide RNA, wherein the guide RNA targets a mutation within a 5 bp BCL11a binding site located in the gamma globin locus on chromosome 11 or one of two SNP mutations in the erythroid-specific enhancer region selected from rs1427407 and rs7569946 located in the BCL11a gene on chromosome 2. See also Fig. 8A , 8B , 34 and 35A-35D.

[0099] In a specific embodiment, a therapeutic nucleic acid sequence (e.g., gene) can be selected to be incorporated into a genetic locus to provide in vivo selection of genetically modified cells. For example, in vivo selection using a cell growth switch allows a minority of genetically modified cells to be induced to expand. A strategy to achieve in vivo selection is to use drug selection while co-expressing a transgene that delivers chemical resistance, such as O6-methylguanine-DNA-methyltransferase MGMT. An alternative approach is to confer enhanced proliferation potential to genetically modified HSCs by delivering the homeobox transcription factor HOXB4. In a specific embodiment, a suicide gene can be incorporated into genetically modified cells, so that such a cell population can be eliminated, for example, by administering a drug that activates the suicide gene. See, for example, Cancer Gene Ther. 2012Aug; 19(8): 523-9; PLoS One. 2013; 8(3): e59594 and Molecular Therapy—Oncolytics (2016) 3, 16011.

[0100] Specific embodiments include contacting blood cells with a gene editing system capable of inserting a donor template at a target site. In specific embodiments, the gene editing system comprises a crRNA capable of hybridizing to a target sequence and a nucleic acid encoding a nuclease such as Cpf1 or Cas9.

[0101] Specific embodiments include contacting blood cells with a gene editing system capable of inserting a donor template at a target site. In specific embodiments, the gene editing system comprises a crRNA capable of hybridizing with a target sequence and a nucleic acid encoding a nuclease such as Cpf1 or Cas9. In specific embodiments, the Cas9 or Cpf1 coding sequence may comprise SEQ ID NOs: 112-124. In specific embodiments, the Cas9 or Cpf1 amino acid sequence may comprise SEQ ID NOs: 125-138.

[0102] (II) Nanoparticles and their conjugation to gene editing components. As noted, there is a need for delivery methods of gene editing systems that do not rely on electroporation, viral vectors, and / or cell selection or purification processes.

[0103] The present disclosure provides engineered NPs that allow gene editing components to be delivered without relying on electroporation or viral vectors to deliver gene editing components. When therapeutic use only requires inactivating problematic genes, NP only needs to be associated with targeting elements and cutting elements (although other components may be included as needed or to contribute to a specific purpose). When therapeutic use adds or corrects genes, NP is associated with targeting elements, cutting elements, and donor templates. In order to further avoid cell selection or purification processes, targeting ligands can be connected to NPs to cause NPs to be selectively delivered to selected cell populations in heterogeneous cell pools.

[0104] Specific embodiments utilize colloidal metal NPs. Colloidal metals include any water-insoluble metal particles or metal compounds dispersed in liquid water. Colloidal metals can be suspensions of metal particles in aqueous solution. Any metal that can be made into colloidal form can be used, including Au, silver, copper, nickel, aluminum, zinc, calcium, platinum, palladium, and iron. In specific embodiments, AuNPs prepared, for example, from HAuCl4 are used. In specific embodiments, NPs are non-Au NPs coated with Au to prepare Au-coated NPs.

[0105] Methods for preparing colloidal metal NPs, including Au colloidal NPs from HAuCl4, are known to those of ordinary skill in the art. For example, the methods described herein and those described elsewhere (e.g., US 2001 / 005581; 2003 / 0118657; and 2003 / 0053983) can be used to prepare NPs.

[0106] In a specific exemplary embodiment, AuNP cores of three different size ranges (15, 50, 100 nm) were synthesized by optimized Turkevich and seed growth methods (Shahbazi, et al., Nanomedicine (Lond), 2017. 12(16): p. 1961-1973; Shahbazi, et al., Nanotechnology, 2017. 28(2): p. 025103; Turkevich, et al., Discussions of the Faraday Society, 1951. 11(0): p. 55-75; Perrault & Chan, Journal of the American Chemical Society, 2009. 131(47): p. 17042-17043). In the first step, 15 nm seed AuNPs were synthesized by bringing 100 mL of 0.25 mM gold (III) chloride trihydrate solution to boiling point and adding 1 mL of 3.33% trisodium citrate dehydrate solution. The synthesis of NPs was carried out under high-speed stirring for 10 min. The prepared NPs were cooled to 40 °C and used for the following growth steps.

[0107] In order to prepare AuNPs of 50nm and 100nm size range, two different 100mL 0.25mM gold (III) chloride trihydrate solutions were prepared, and 2440 μL and 304 μL seed AuNPs were added under gentle stirring conditions to synthesize 50nm and 100nm AuNPs respectively. 1mL 15mM trisodium citrate dehydrate solution was added to these solutions, and the mixture was allowed to reach the highest stirring speed. Then, 1mL 25mM hydroquinone solution was added, and the synthesis lasted 30 minutes for 50nm AuNPs, and 5 hours for 100nm AuNPs. Finally, the synthesized NPs were purified by centrifugation at 5000xg and dispersed in ultrapure water. In a specific embodiment, the NP core>100nm;>90nm;>80nm;>70nm;>60nm;>50nm;>40nm;>30nm; or 20nm.

[0108] Although AuNPs are specifically described, the NPs included in the present disclosure can be provided in different forms, such as solid NPs (e.g., metals such as silver, Au, iron, titanium), non-metals, lipid-based solids, polymers, suspensions of NPs, or combinations thereof. Metallic, dielectric, and semiconductor NPs can be prepared, as well as hybrid structures (e.g., core-shell NPs). NPs made of semiconductor materials can also be labeled as quantum dots, and if they are small enough (usually less than 10 nm), quantization of electronic energy levels occurs. Such nanoscale particles are used as drug carriers or imaging agents in biomedical applications, and can be suitable for similar purposes in the present disclosure.

[0109] As noted, a variety of active components can be conjugated to the NPs disclosed herein for targeted gene editing. For example, nucleic acids as components of a gene editing system can be directly or indirectly and covalently or non-covalently conjugated to the surface of the NP. For example, the nucleic acid can be covalently bound to the surface of the NP at one end of the nucleic acid.

[0110] The nucleic acid conjugated to NP can have a length of 10 nucleotides (nt)-1000nt, such as 1nt-25nt, 25nt-50nt, 50nt-100nt, 100nt-250nt, 250nt-500nt, 500nt-1000nt or greater than 1000nt. In a specific embodiment, the length of the nucleic acid modified by conjugation with a linker does not exceed 50nt or 40nt.

[0111] When conjugated indirectly, for example, by an intermediate linker, any type of molecule can be used as a linker. For example, the linker can be an aliphatic chain comprising at least two carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more carbon atoms) and can be substituted with one or more functional groups, including ketones, ethers, esters, amides, alcohols, amines, ureas, thioureas, sulfoxides, sulfones, sulfonamides and / or disulfides.

[0112] In a specific embodiment, the linker comprises a disulfide at the free end (e.g., the end not conjugated to the guide RNA) of the coupled NP surface. In a specific embodiment, the disulfide is a C2-C10 disulfide, that is, it can be a disulfide-terminated aliphatic chain including 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, although it is envisioned that longer aliphatic chains can be used. In a specific embodiment, the disulfide is a 3-carbon disulfide (C3 SS). The linker can have a sulfhydryl (SH) or a disulfide (SS) or a different number of sulfur atoms. In a specific embodiment, a thiol modification can be introduced without the use of a linker. In a specific embodiment, the nuclease is delivered as a protein (ribonucleoprotein (RNP) complex) pre-conjugated with its guide RNA. In this formulation, the guide RNA molecule is bound to the NP, and by default, the nuclease can also be bound (see, e.g., Figure 5B ).

[0113] An improvement disclosed herein is the ability to modify CRISPR components to be connected to NP. This is because most modifications in CRISPR components may impair cutting efficiency. For example, Li et al. (Engineering CRISPR-Cpf1 crRNAs andmRNAs to maximize genome editing efficiency.2017.1: Page 0066) show that the 5' end of Cpf1 crRNA is unsafe for any modification, because such modifications lead to the elimination of the binding of crRNA to Cpf1 nuclease. Modification of the 3' end of crRNA without impairing cutting efficiency is disclosed herein. In a specific embodiment, in the first step of conjugation to NP, the 3' end of crRNA is modified with 18-atom hexaethylene glycol spacers (18 spacers) and 3 carbon disulfides (C3S-S) to connect crRNA to the surface of AuNP.

[0114] Based on the foregoing, in a specific embodiment, for example, when NP includes Au, the linker can be any molecule containing thiol. The reaction of thiol group with Au produces covalent sulfide (-S-) bond. AuNP has high affinity for thiol (-SH) and dithiol (SS) groups, and there is a semi-covalent bond between the surface of AuNP and the sulfide group (Hakkinen, Nat Chem, 2012.4 (6): pp. 443-455). In a specific embodiment, thiol groups can be added to nucleic acids to promote connection with the surface of AuNP. This method can improve nucleic acid uptake and stability (see, for example, Mirkin, et al., A Nature, 1996.382 (6592): pp. 607-609).

[0115] Highly monodisperse AuNPs were synthesized in three different size ranges (15 nm, 50 nm, 100 nm) using an optimized two-step seeded growth method and conjugated with Cpf1 crRNA and endonuclease ( Figure 5B and 11B ). Due to the strong electrostatic repulsion between the negatively charged surface and the negatively charged crRNA, it is difficult to connect crRNA to the surface of AuNP without, for example, thiol modification. In a specific embodiment, in a second step, after purifying the crRNA-conjugated AuNP, Cpf1 endonuclease is added and incubated with the crRNA-conjugated AuNP to promote its binding to the 5' handle of crRNA (Dong, et al., Nature, 2016.532(7600): pp. 522-526), ​​the compact structure of the designed NP containing both crRNA and Cpf1 endonuclease leads to the following conformation, which increases the stability to the degrading agent and promotes the uptake of Au / CRISPR NP by cells due to the overall neutral charge (i.e., zeta potential). Although the disclosed NPs are given special relevance for CRISPR / Cpf1 optimization, the same concept can be applied to other CRISPR categories. Furthermore, together with crRNA and Cpf1 endonuclease, 18-spacer thiol-modified single-stranded DNA (ssDNA) can be attached to the surface of AuNPs to obtain a new type of NPs with the purpose of homology-directed repair (HDR).

[0116] In a specific embodiment, a spacer-thiol linker can be added to Cpf1 or Cas9 protein itself or the aforementioned engineered variant (for example, as described below) by adding a cysteine ​​residue at the N- or C-terminus. The nuclease protein can then be added to the surface of the AuNP core as the first layer. This spacer-thiol linker can increase the stability of the protein and increase the cutting efficiency. In a specific embodiment, the RNA complex is formed between crRNA and the nuclease, and then connected to the surface of the AuNP core surface by a spacer-thiol linker.

[0117] As previously described, adding bacterially derived gene editing components as the first loading step can provide beneficial shielding of these components after administration to subjects with pre-existing immunity to the components. Shielding can be attributed to other gene editing components (e.g., donor template) and does not need to rely on a protective polymer shell. In specific embodiments, a polymer shell is not included. In specific embodiments, shielding allows for continuous in vivo administration.

[0118] In a specific embodiment, crRNA can be added to AuNPs with different AuNP / crRNA w / w ratios (0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6) and mixed. A citrate buffer with a pH of 3 can be added to the mixture at a concentration of 10 mM to screen for negative repulsion between negatively charged crRNA and AuNP. After stirring for 5 minutes, NPs were centrifuged and unbound crRNA was displayed by agarose gel electrophoresis. After determining the optimal conjugation concentration, 1 μL 63 μM Cpf1 nuclease can be added to the AuNP / crRNA solution and incubated for 20 minutes.

[0119] Importantly, the use of citrate buffer provides significant advantages in manufacturing. Previous methods rely on the use of NaCl to screen negatively charged NP surfaces and reduce the repulsion of similar negatively charged DNA. However, NaCl can cause irreversible aggregation of AuNPs, so it must be gradually added over time as the concentration increases. Typically, NaCl must be added within 48 hours to avoid aggregation. When using a citrate buffer with a pH of 3, this combination can occur with higher efficiency in less than 3 minutes. Zhang, et al. (2012). Journal of the American Chemical Society 134 (17): 7266-7269, reducing product costs and time in GMP manufacturing plants.

[0120] The size and morphology of the prepared Au / CRISPRNPs can be characterized by imaging under a transmission electron microscope (TEM). AuNPs (4 μL) can be added to a copper grid and allowed to dry overnight. Imaging was performed at 120 kV.

[0121] Coatings with gene editing components can be visualized by negative staining electron microscopy. For example, NPs can be stained with 0.7% uranyl formate and 2% uranyl acetate, respectively. The stained sample (4 μL) can be added to a carbon-coated copper grid and incubated for 1 minute and blotted with filter paper. After three wash cycles with 20 μl of dye solution, 4 μl of dye solution can be added to the grid, blotted and air-dried.

[0122] The NPs can also be characterized by a Nanodrop UV-visible spectrophotometer by analyzing the shift of the localized surface plasmon resonance (LSPR) peak of the NPs before and after conjugation with the gene editing components.

[0123] In certain embodiments, NPs are layered, such as during synthesis, to include PEI or other positively charged polymers for increasing surface area and conjugating larger ssDNA or other molecules, such as targeting ligands and / or large donor templates (see, e.g., Figure 6B ). Such NPs can be prepared in a layer-by-layer format, and positively charged polymers (such as; PEI of different molecular weights and forms) can be used to coat the negatively charged surface of AuNPs or AuNPs coated with gene editing components to connect gene editing components and other components (such as antibody binding domains). Layering substantially increases the surface area of ​​NPs available for conjugation of molecules (such as large oligonucleotides with or without other proteins).

[0124] Particular embodiments utilize positively charged polymers having a molecular weight of 1,000-3,000 Daltons (e.g., 1,000; 1,200; 1,400; 1,600; 1,800; 2,000; 2,200; 2,400; 2,600; 2,800; or 3,000 Daltons). Examples of positively charged polymers include polyamines; polyorganoamines (e.g., polyethyleneimine (PEI), polyethyleneimine cellulose); poly(amidoamine) (PAMAM); polyamino acids (e.g., polylysine (PLL), polyarginine); polysaccharides (e.g., cellulose, dextran, DEAE dextran, starch); spermine, spermidine, poly(vinylbenzyltrialkylammonium), poly(4-vinyl-N-alkyl-pyridinium), poly(acryloyl-trialkylammonium), and Tat proteins.

[0125] Blends of polymers (and optional lipids) at any concentration and in any ratio may also be used. Blending different polymer types using various grades in different ratios may produce features borrowed from each contributing polymer. Various end group chemistries may also be employed.

[0126] In a particular embodiment, a positively charged polymer (e.g., PEI) can be added as a coating onto an already formed portion of the NP, and the ssDNA can be added simultaneously or subsequently. Alternatively, the conjugation step can be altered by adding the ssDNA as a layer and then adding the positively charged polymer as a subsequent layer. In a particular embodiment, a positively charged polymer and ssDNA are not included as the first layer, as this layer can be reserved for the RNP complex coupled to the linker.

[0127] In certain embodiments, the multilayer NPs of the present disclosure have an average size of 25-70 nm and are highly monodisperse. Transmission electron microscopy images (TEM) and LSPR of AuNPs show a uniform surface coating without any aggregation ( Fig. 10A , 10B Given the synthetic nature of the entire delivery system, all components can be assembled within a few hours, in stark contrast to previous approaches that required multiple days due to, for example, the use of NaCl as a charge screen.

[0128] like Fig. 10AAs shown, the synthesized NPs were highly monodisperse and a successful 4 nm coating was achieved without any aggregation, which increased the size of the NPs to 54 nm after coating with 50 nm AuNPs. In addition, the decrease in the intensity and red shift of the LSPR of AuNPs indicated successful conjugation with the gene editing components without any aggregation ( Fig. 10A ). Each layer will have a different optimal loading ratio. The first layer consists of RNA, however, to test the optimal loading ratio of this layer, a single-stranded DNA test nucleotide (ssDNA) was used. This test oligonucleotide was modified using the same 18-spacer C3 SS used to modify crRNA. In the loading study, different AuNP / crRNA w / w ratios showed that a ratio of 6 for particle core:ssDNA (and, by inference, crRNA) was optimal for conjugation ( Fig. 10C Using this optimal loading ratio, crRNA was loaded on the surface of AuNPs at a concentration of 30 μg / mL ( Fig. 10D ). These data help calculate the precise dosage to be applied in gene editing research.

[0129] As will be appreciated by one of ordinary skill in the art, the ratios provided are iterative, as the ratios for optimal loading are slightly different as each layer is added. The characteristics of the NP as a whole, as well as the last layer added, and the characteristics of the new layer to be added all affect the ratios. In a specific embodiment, a ratio of 6:1 is optimal for crRNA (first layer). In a specific embodiment, for Cpf1 protein, a ratio of 0.6 is optimal for loading onto the NP core+crRNA layer, and the final HDT layer has an optimal loading ratio of 1. Modifications or changes in length of the Cpf1 protein or chemical modifications of the HDT can affect these ratios.

[0130] Particularly useful ratios of particle core to gene editing components include a weight / weight (w / w) ratio of 0.5; 0.6; or 0.7 for particle core:Cpf1; a weight / weight (w / w) ratio of 0.9; 1.0; or 1.1 for particle core:HDT.

[0131] The described methods produce highly efficient, loaded, gene-edited NPs capable of delivering synthetic, non-chemically modified ribonucleoproteins as well as ssDNA homologous templates to insert new DNA without the need for electroporation or viral vector delivery. In specific embodiments, the hydrodynamic size of the fully loaded AuNPs is 150-190nm, 160-185nm, 170-180nm, or 176nm.

[0132] Additional particle designs include the following components extending from the proximal end to the distal end of the NP core surface in the following order: thiolated PEI, a linker, a targeting element, and a cutting element. In a specific embodiment, the linker is a polyethylene glycol linker. In a specific embodiment, a water-soluble amine-thiol crosslinker containing NHS-ester and maleimide reactive groups at the opposite ends of a medium-length cyclohexane spacer arm can be used to connect the cutting element to the targeting ligand. In a specific embodiment, the amine-thiol crosslinker includes sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate (sulfo-SMCC, Fig. 6E ). In a particular embodiment, the ssDNA is in a layer surrounding the NP core that is coextensive with the layer of linkers. This configuration is, for example, Figure 5D and 6C -depicted in 6E.

[0133] The joint includes a polymer joint. In a specific embodiment, the joint can be an amino acid sequence with 1 to 500 amino acids, which can provide flexibility and space for the conformational movement between two regions, domains, motifs, boxes or modules connected by a joint. In a specific embodiment, the joint can be flexible, rigid or semi-rigid, depending on the desired function or structure of the component connected by the joint. In a specific embodiment, when a joint connects two molecules, regions, domains, motifs, boxes or modules, the joint can be direct. In a specific embodiment, when two molecules, regions, domains, motifs, boxes or modules are not directly connected by a single joint but are connected to a third joint or domain from both sides by a joint, the joint can be indirect. Exemplary joint sequences include one to ten Gly x Ser y A repeated linker sequence, wherein x and y are independently an integer from 0 to 10, provided that x and y are not simultaneously 0 (e.g., ((Gly4Ser)3(SEQ ID NO:98), (Gly3Ser)2(SEQ ID NO:99), Gly2Ser or a combination thereof, such as (Gly3Ser)2Gly2Ser) (SEQ ID NO:100)).

[0134] Examples of rigid or semi-rigid linkers include proline-rich linkers. In specific embodiments, a proline-rich linker is a peptide sequence having more proline residues than would be expected based solely on chance. In specific embodiments, a proline-rich linker is a linker having at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% proline residues. Specific examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).

[0135] (III) Gene editing efficiency. The optimal concentrations of crRNA, hAsCpf1 RNA, and ssODN for electroporation were determined in K562 cells. The optimal concentration showed the highest viability and GFP expression. 5 K562 cells were cultured at a concentration of 10 cells / well. The cells were cultured in Iscove's modified Dulbecco's medium (IMDM) containing 10% FBS and 1% PenStrep. 5 × 10 5 CD34+ cells are cultured at a concentration of 10 cells / well. The culture conditions of CD34+ cells are the same as K562 cells with the required growth factors. Au / CRISPR NPs are added to these wells at a concentration of 25nM and the editing efficiency is evaluated after 48 hours of incubation. In specific embodiments, AuNP / CRISPR can be incubated with the cell population for 1-48 hours, 1-36 hours, 1-24 hours, or 1-12 hours. In certain embodiments, AuNP / CRISPR can be incubated with a population of cells for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours or more. Electroporation of cells was performed using BTX Express Solution (USA) in 1mm cuvettes at 250V and 5ms pulse duration using Harvard Apparatus ECM 830 square wave electroporation system. 1-3 million K562 cells were electroporated for 5 milliseconds at 250V using a 1mm BTX cuvette with a 2mm gap width. Cells were resuspended in culture medium and analyzed after electroporation. In the case of minimal operation embodiments, 1-24, 1-48 or 1-72 hours are preferred for clinical logic or disease conditions. In some cases, it may take 2 days to adjust cancer patients for reinfusion, but in the case of genetic diseases, patients may not be adjusted and preferably limit the in vitro operation time.

[0136] Compared with the electroporation method using higher amounts of crRNA and Cpf1 (126 nM), AuNP / CRISPR targeting chr11:67812349-67812375 was able to successfully cleave the target site with very low crRNA and Cpf1 endonuclease concentrations (25 nM). Fig. 16C ) to achieve the same cleavage efficiency. Due to the A>T mutation 15bp after the PAM site, the cleavage efficiency at this site was very low. In the next test, the same position was targeted in primary CD34+ cells and it was shown that Au / CRISPR NPs were able to target the site with very good cleavage efficiency at very low crRNA and Cpf1 endonuclease concentrations without any toxic effects ( Fig.16A , 16D and 18). Unfortunately, electroporation of primary CD34+ cells adversely affected cell viability, and no cleavage was observed for electroporated cells. The calculated concentration of AuNP / CRISPR was 5-fold lower than that required for the electroporation method ( Fig. 16B As previously described by Kim et al. (Nat Biotechnol, 2016.34(8): p. 863-8), the CRISPR Cpf1 gene editing system has a higher ratio of deletions to insertions ( Fig.18 ).

[0137] like Figures 23A-23C As shown, AuNP-mediated gene delivery improves Cas9 performance, however, Cpf1 is better for HDR. Compared with electroporated cells, AuNP-treated cells show higher viability. For Cas9, AuNP-mediated delivery improves total editing and HDR relative to electroporation. For Cpf1 not delivered using homology-directed repair template (HDT), electroporation leads to higher total gene editing (insertions and deletions, indels). This shows that electroporation itself may affect the repair pathway used or the frequency of Cpf1 cutting at the target site. Adding HDT to Cpf1 preparations improves total editing and obtains the highest HDR rate. In short, these data show that the fully loaded preparation of AuNP+Cpf1 / crRNA+HDT obtains the highest HDR rate and the least indel formation. This is ideal for many target loci for gene editing.

[0138] In a specific embodiment, many assays known in the art can be used to detect gene editing and / or gene editing levels (percentages) or gene editing rates. In a specific embodiment, the deletion or introduction of enzyme restriction sites as a result of gene editing can be evaluated by restriction enzyme digestion of amplified genomic DNA flanking the gene editing target site and observing the digestion products by gel electrophoresis. In a specific embodiment, T7 endonuclease I (T7EI) assay can be used. In the T7EI assay, genomic DNA can be isolated from cells that have been targeted for genetic modification, and genomic regions flanking the gene editing target site can be PCR amplified. The amplified product can be annealed and digested with T7EI. T7EI recognizes and cleaves non-completely matched DNA, so any gene editing can be detected as a mismatch in the annealed heteroduplex, which is then cut by T7EI. The percentage of gene modification in the T7EI assay can be calculated as follows: Percentage of gene modification = 100x (1-(1-cleavage portion) 1 / 2 ). The T7EI assay kit can be obtained, for example, from New England Biolabs, Ipswich, MA.

[0139] In a specific embodiment, gene editing or gene editing level (percentage) can be detected by decomposition tracking insertion and deletion (TIDE) assay. The genomic region flanking the gene editing target site can be PCR amplified, and the amplified product can be purified. Sanger sequencing of the purified product can be performed with fluorescently labeled termination dideoxynucleoside triphosphates (sequencing kits can be purchased from, for example, Thermo Fisher Scientific, Waltham, MA). After cycle sequencing, the obtained sequence can be run on TIDE software. The results can be reported as a percentage of gene modification (Brinkman et al., Nucleic Acids Research, 42 (22): e168-e168 (2014)).

[0140] In a specific embodiment, gene editing or the level (percentage) of gene editing can be detected by sequencing. The genomic region flanking the gene editing target site can be PCR amplified, and the amplified product can be purified. A second PCR can be performed to add the adapter and / or other sequences required for a given sequencing platform. Any sequencing method can be used, including synthetic sequencing, pyrophosphate sequencing, connection sequencing, rolling circle amplification sequencing, single molecule real-time sequencing, sequencing based on release proton detection, and nanopore sequencing.

[0141] In certain embodiments, the use of therapeutic formulations comprising the NPs described herein can produce an average of 5% to 100%, 5% to 90%, 5% to 80%, 5% to 70%, 5% to 60%, 5% to 50%, 5% to 40%, 5% to 30%, or 5% to 20% total gene editing in target cells. In certain embodiments, the use of therapeutic formulations comprising the NPs described herein can produce an average of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more total gene editing in target cells.

[0142] Confocal microscopy showed that the disclosed NPs avoided lysosomal entrapment and successfully localized to the nuclei of CD34+ primary hematopoietic cells from healthy donors. Using NotI restriction enzyme templates with homology arm lengths of ±40 nucleotides to the CCR5 locus showed up to 10% knock-in frequency without cytotoxicity. Designing templates for non-target DNA strands resulted in higher homology-directed repair (HDR) efficiency ( Fig.17 ), with clear cleavage bands of 447 bp and 316 bp after digestion with NotI and T7EI enzymes ( Fig.19B ). Direct comparison of Cpf1 and Cas9 nuclease activities at the same CCR5 target site demonstrated that Cpf1 favors HDR and template knock-in relative to Cas9, which preferentially generates indels. Xenotransplantation of CRISPR Cpf1NP-treated human CD34+ cells into immunodeficient mice showed a trend toward an early increase in engraftment compared to untreated cells, suggesting an unknown benefit of NP-treated HSPCs. The frequency of CCR5 genetically modified cells engraftment was identical to that observed in culture, with 10% of human cells showing Notl template addition in vivo.

[0143] In a specific embodiment, during the incubation period, 1, 2, 3, 4, 5, 8, 10, 12, 15 or 20 μg / mL NPs are added per mL of the least manipulated blood cell product. The incubation period can be, for example, 40 minutes to 48 hours long (in a specific embodiment, 1 hour). In a specific embodiment, the incubation time is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and each integer of up to 48 hours. Incubation can be carried out at 2-8 degrees Celsius (refrigerated), 23-28 degrees Celsius (room temperature) or 37 degrees Celsius (body temperature). During the incubation process at any temperature, slight shaking or rotation of the product may occur.

[0144] (IV) selected cells and selected cell targeting ligands. The cell population (i.e., cell type) to be targeted for genetic modification includes HSC, HSPC, hematopoietic progenitor cells (HPC), T cells, B cells, natural killer (NK) cells, macrophages, monocytes, mesenchymal stem cells (MSC), white blood cells (WBC), mononuclear cells (MNC), endothelial cells (EC), stromal cells and / or bone marrow fibroblasts. The selected cell population may refer to a cell population that is targeted by the NP of the present invention or has been targeted by the NP of the present disclosure for genetic modification.

[0145] HSC is multipotent and eventually produces all types of terminally differentiated blood cells.HSC can self-renew, or it can differentiate into more stereotyped progenitor cells, which are irreversibly determined as the ancestors of only a few types of blood cells.For example, HSC can differentiate into (i) bone marrow progenitor cells, which eventually produce monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells, or (ii) lymphoid progenitor cells, which eventually produce T cells, B cells and NK cells.Once stem cell differentiation becomes bone marrow progenitor cells, its offspring can not produce lymphoid lineage cells, and similarly, lymphoid progenitor cells can not produce bone marrow lineage cells. For a general discussion of hematopoiesis and hematopoietic stem cell differentiation, see Differentiated Cells and the Maintenance of Tissues, Chapter 17, Alberts et al., 1989, Molecular Biology of the Cell, 2nd ed., Garland Publishing, New York, NY; Regenerative Medicine, Department of Health and Human Services, Chapter 2, August 2006, and Hematopoietic Stem Cells, Chapter 5, 2009, Stem Cell Information, Department of Health and Human Services.

[0146] Specific HSC populations include HSC1 (Lin-CD34+CD38-CD45RA-CD90+CD49f+) and HSC2 (CD34+CD38-CD45RA-CD90-CD49f+). For example, in a specific embodiment, human HSC1 can be identified by the following profile: CD34+ / CD38- / CD45RA- / CD90+ or ​​CD34+ / CD45RA- / CD90+, and mouse LT-HSC can be identified by Lin-Sca1+ckit+CD150+CD48-Flt3-CD34- (wherein Lin represents any marker that does not express mature cells, including CD3, Cd4, CD8, CD11b, CD11c, NK1.1, Gr1 and TER119). Therefore, HSC1 can include a marker profile: LH R+ / CD34+ / CD38- / CD45RA- / CD90+. In addition to the expression of LHR, in certain embodiments, HSC1 can be identified by the following profile: Lin- / CD34+ / CD38- / CD45RA- / CD90+ / CD49f+. Thus, HSC1 can include a marker profile: LHR+ / Lin- / CD34+ / CD38- / CD45RA- / CD90+ / CD49f+. In addition to the expression of LHR, in certain embodiments, HSC2 can be identified by the following profile: CD34+ / CD38- / CD45RA- / CD90- / CD49f+. Thus, HSC2 can include a marker profile: LHR+ / CD34+ / CD38- / CD45RA- / CD90- / CD49f+. Based on the foregoing profiles, the expression of LHR can be combined with the presence or absence of one or more of the following markers to identify HSC1 and / or HSC2 cell populations: Lin / CD34 / CD38 / CD45RA / CD90 / CD49f and CD133. Various other combinations can also be used, as long as the marker combination reliably identifies HSC1 or HSC2. In a specific embodiment, HSC is identified by a CD133+ profile. In a specific embodiment, HSC is identified by a CD34+ / CD133+ profile. In a specific embodiment, HSC is identified by a CD164+ profile. In a specific embodiment, HSC is identified by a CD34+ / CD164+ profile.

[0147] HSPC refers to hematopoietic stem cells and / or hematopoietic progenitor cells. HSPC can self-renew or can differentiate into bone marrow progenitor cells or lymphoid progenitor cells, as described above for HSC. Relative to other types of hematopoietic cells, HSPC can be positive for specific markers expressed at increased levels on HSPC. For example, such markers include CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR or a combination thereof. Moreover, relative to other types of hematopoietic cells, HSPC can be negative for markers expressed. For example, such markers include Lin, CD38 or a combination thereof. Preferably, HSPC is a CD34+ cell.

[0148] In certain embodiments, 'HSC / HSPC' may refer to HSC, HSPC, or both.

[0149] Lymphocytes include T cells and B cells. T cells are a key part of the immune system, helping to control immune responses and kill cells, such as virus-infected cells and cancer cells. There are several types of T cells, including helper T cells, cytotoxic T cells, central memory T cells, effector memory T cells, regulatory T cells, and naive T cells. B cells are involved in the adaptive immune system, including the production of antibodies against invaders such as bacteria, viruses, and other organisms.

[0150] Several different T cell subsets have been discovered, each with different functions. In a specific embodiment, the selected cell targeting ligand achieves selective targeting to a specific lymphocyte population by receptor-mediated endocytosis. For example, most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor consists of two separate peptide chains, which are produced by independent T cell receptor α and β (TCRα and TCRβ) genes and are called α- and β-TCR chains.

[0151] γδ T cells represent a small subset of T cells that have a different T cell receptor (TCR) on their surface. In γδ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells is much less common than αβ T cells (2% of total T cells).

[0152] CD3 is expressed on all mature T cells. Therefore, the selected cell targeting ligand disclosed herein can bind CD3 to achieve selective delivery of nucleic acid to all mature T cells. Activated T cells express 4-1BB (CD137), CD69 and CD25. Therefore, the selected cell targeting ligand disclosed herein can bind 4-1BB, CD69 or CD25 to achieve selective delivery of nucleic acid to activated T cells. CD5 and transferrin receptor are also expressed on T cells.

[0153] T cells can be further divided into helper cells (CD4+T cells) and cytotoxic T cells (CTL, CD8+T cells), which include cytolytic T cells. T helper cells assist other white blood cells in the immune process, including maturing B cells into plasma cells and activating cytotoxic T cells and macrophages. These cells are also called CD4+T cells because they express CD4 proteins on their surfaces. When helper T cells are presented with peptide antigens by MHC class II molecules expressed on the surface of antigen presenting cells (APCs), they are activated. Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist active immune responses.

[0154] Cytotoxic T cells destroy virus-infected cells and tumor cells, and are also involved in transplant rejection. These cells are also called CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost every cell in the body.

[0155] As used herein, "central memory" T cells (or "TCMs") refer to antigen-experienced CTLs that express CD62L or CCR7 and CD45RO on their surface, and do not express CD45RA or have reduced CD45RA expression compared to naive cells. In specific embodiments, central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have reduced CD45RA expression compared to naive cells.

[0156] As used herein, "effector memory" T cells (or "TEMs") refer to antigen-experienced T cells that do not express CD62L or have reduced CD62L expression on their surface compared to central memory cells, and do not express CD45RA or have reduced CD45RA expression compared to naive cells. In a specific embodiment, effector memory cells are negative for expression of CD62L and CCR7 compared to naive cells or central memory cells, and have variable expression of CD28 and CD45RA. Effector T cells are positive for granzyme B and perforin compared to memory or naive T cells.

[0157] Regulatory T cells ("TREG") are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens and eliminate autoimmune diseases. TREG express CD25, CTLA-4, GITR, GARP and LAP.

[0158] As used herein, "naive" T cells refer to antigen-naive T cells that express CD62L and CD45RA, and do not express CD45RO in contrast to central or effector memory cells. In a specific embodiment, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T cells, including CD62L, CCR7, CD28, CD127, and CD45RA.

[0159] B cells can be distinguished from other lymphocytes by the presence of B cell receptors (BCR). The main function of B cells is to produce antibodies. B cells express CD5, CD19, CD20, CD21, CD22, CD35, CD40, CD52 and CD80, and the selected cell targeting ligands disclosed herein can bind CD5, CD19, CD20, CD21, CD22, CD35, CD40, CD52 and / or CD80 to achieve selective delivery of nucleic acids to B cells. Antibodies targeting the constant region of the B cell receptor isotype (IgM, IgG, IgA, IgE) can also be used to target B cell subtypes.

[0160] Natural killer cells (also known as NK cells, K cells and killer cells) are activated in response to interferon or macrophage-derived cytokines. NK cells can induce apoptosis or cell lysis by releasing particles that destroy the cell membrane, and can secrete cytokines to recruit other immune cells. They are used to contain viral infections, while adaptive immune responses produce antigen-specific cytotoxic T cells that can clear infections. NK cells express NKG2D, CD8, CD16, CD56, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D and several members of the natural cytotoxicity receptor (NCR) family. Examples of NCRs include NKp30, NKp44, NKp46, NKp80 and DNAM-1.

[0161] Macrophages (and their precursors, monocytes) are present in every tissue of the body (as are microglia, Kupffer cells, and osteoclasts in some cases), where they engulf apoptotic cells, pathogens, and other non-self components. Examples of proteins expressed on the surface of macrophages (and their precursors, monocytes) include CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4 / 80, IFGR2, Toll-like receptors (TLR) 1-9, IL-4Rα, and MARCO.

[0162] The selected cell targeting ligands that can be connected to the NP disclosed herein selectively bind to target cells within a heterogeneous cell population. "Selective delivery" to a selected cell type within a heterogeneous cell mixture refers to at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of the administered NPs being proportionally taken up by the targeted cells relative to cells in the population that do not express the target marker. In a specific embodiment, 50% or more of the selected cell population in the sample takes up the NPs, and less than 20% of any non-target cell population takes up the NPs.

[0163] In certain embodiments, the binding domain of the selected cell targeting ligand comprises a cell marker ligand, a receptor ligand, an antibody, a peptide, a peptide aptamer, a nucleic acid, a nucleic acid aptamer, a spiegelmer, or a combination thereof. In the case of the selected cell targeting ligand, the binding domain comprises any substance that binds to another substance to form a complex capable of mediating endocytosis.

[0164] "Antibodies" are an example of targeting ligands and include whole antibodies or binding fragments of antibodies, such as Fv, Fab, Fab', F(ab')2 and single-chain Fv fragments (scFv) or any biologically effective fragment of an immunoglobulin that specifically binds to a motif expressed by a selected cell. Antibodies or antigen-binding fragments include all or part of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, minibodies and linear antibodies.

[0165] A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of an immunoglobulin connected by a short linker peptide. L and V H domain, but lacks the constant region. Although the two domains V L and V H are encoded by separate genes, but they can be joined by a synthetic linker that enables them to be made into a single protein chain using, for example, recombinant methods, where V L and V HThe regions are paired to form a monovalent molecule (single-chain Fv (scFv)). For additional information on Fv and scFv, see, for example, Bird, et al., Science 242 (1988) 423-426; Huston, et al., Proc. Natl. Acad. Sci. USA 85 (1988) 5879-5883; Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York), (1994) 269-315; WO 1993 / 16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.

[0166] The Fab fragment is composed of V L 、V H , CL and CH1 domains. F(ab')2 fragments are bivalent fragments comprising two Fab fragments connected by a disulfide bond at the hinge region. Diabodies include two epitope binding sites that can be bivalent. See, for example, EP 0404097; WO1993 / 01161; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448. Dual affinity retargeting antibodies (DARTs) can also be formed. TM ; Based on the diabody format, but characterized by a C-terminal disulfide bond for additional stabilization (Moore et al., Blood 117, 4542-51 (2011)). Antibody fragments can also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat Med. 9 (2003) 129-134.

[0167] Antibodies from human sources or humanized antibodies have reduced or no immunogenicity in humans and have a lower number of non-immunogenic epitopes compared to non-human antibodies. Antibodies and fragments thereof are typically selected to have reduced levels of antigenicity or no antigenicity in human subjects.

[0168] Methods for obtaining monoclonal antibodies, methods for phage display, methods for producing human or humanized antibodies, or methods using transgenic animals or plants engineered to produce antibodies can be used to prepare antibodies that specifically bind to motifs expressed by selected cell types, as known to those of ordinary skill in the art (see, e.g., U.S. Pat. Nos. 6,291,161 and 6,291,158), phage display libraries of partially or completely synthetic antibodies can be obtained, and antibodies or fragments thereof that can bind to the motifs of the selected cell type can be screened. For example, binding domains can be identified by screening Fab phage libraries for Fab fragments that specifically bind to the target of interest (see Hoet et al., Nat. Biotechnol. 23:344, 2005). Phage display libraries of human antibodies can also be obtained. In addition, phage display libraries of human antibodies can be used in convenient systems (e.g., mice, HuMAbs, TCmouse TM , Targeted ligand binding domains are developed using traditional strategies for hybridoma development using a target of interest as an immunogen in selected lymphocytes (e.g., llamas, chickens, rats, hamsters, rabbits, etc.). In specific embodiments, the antibody specifically binds to a motif expressed by the selected lymphocytes and does not cross-react with non-specific components or unrelated targets. Once identified, the amino acid sequence or nucleic acid sequence encoding the antibody can be isolated and / or determined.

[0169] Aptamers can be designed to facilitate selective delivery, including delivery across the cell membrane to intracellular compartments or into the nucleus. Methods for preparing aptamers and conjugating such aptamers to the surface of NPs are described, for example, in Huang et al. Anal. Chem., 2008, 80 (3), pp. 567-572. In a specific embodiment, the aptamers of the present disclosure bind CD133.

[0170] In a specific embodiment, a peptide aptamer refers to a peptide loop (which is specific to a target protein) connected to a protein scaffold at both ends. This dual structural constraint greatly increases the binding affinity of a peptide aptamer to a level comparable to that of an antibody. The variable loop length is typically 8 to 20 amino acids (e.g., 8 to 12 amino acids), and the scaffold can be any protein that is stable, soluble, small and non-toxic (e.g., thioredoxin-A, stefin A triple mutant, green fluorescent protein, leech inhibitor (eglin) C and cell transcription factor SpI). Peptide aptamer selection can be performed using different systems, such as a yeast two-hybrid system (e.g., a Gal4 yeast two-hybrid system) or a LexA interaction capture system.

[0171] Nucleic acid aptamers are single-stranded nucleic acid (DNA or RNA) ligands that function by folding into specific globular structures that determine binding with high affinity and specificity to a target protein or other molecule, as described in Osborne et al., Curr. Opin. Chem. Biol. 1:5-9, 1997; and Cerchia et al., FEBS Letters 528:12-16, 2002. In specific embodiments, aptamers are small (15 KD; or between 15-80 nucleotides or between 20-50 nucleotides). Aptamers are typically generated from a population of 10 aptamers by a procedure known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment; see, e.g., Tuerk et al., Science, 249:505-510, 1990; Green et al., Methods Enzymology. 75-86, 1991; and Gold et al., Annu. Rev. Biochem., 64:763-797, 1995). 14 -10 15 aptamers are isolated from a library of random oligonucleotide sequences. Other methods for producing aptamers are described, for example, in U.S. Pat. Nos. 6,344,318; 6,331,398; 6,110,900; 5,817,785; 5,756,291; 5,696,249; 5,670,637; 5,637,461; 5,595,877; 5,527,894; 5,496,938; 5,475,096; and 5,270,16. Spiegelmers are similar to nucleic acid aptamers, except that at least one β-ribose unit is replaced by β-D-deoxyribose or a modified sugar unit selected from, for example, β-D-ribose, α-D-ribose, β-L-ribose.

[0172] In a specific embodiment, the RNA aptamer sequence has binding affinity to the aptamer ligand on or in the cell. In a specific embodiment, the aptamer ligand is on the cell, such as to make it at least partially available for the cell outside or side of the cell membrane. For example, the aptamer ligand can be a cell surface protein. Therefore, the aptamer ligand can be a part of a fusion protein, and the other part of the fusion protein has a membrane anchor or a transmembrane domain. In a specific embodiment, the aptamer ligand is in a cell. For example, the aptamer ligand can be internalized into the cell, i.e., in the cell membrane (beyond the cell membrane), such as in the cytoplasm, in the organelle (including mitochondria), in the endosome or in the nucleus. In a specific embodiment, the aptamer can include a donor template sequence, which can include a homology-directed repair (HDR) template and a therapeutic nucleic acid sequence.

[0173] The selected cell targeting ligand disclosed herein can bind to CD34, CD46, CD90, CD133, CD164, Sca-1, CD117, LHRH receptor and / or AHR to achieve selective delivery of NP to HSC. As previously described, specific embodiments include one or more of CD34 antibody, CD90 antibody, CD133 antibody, CD164 antibody, aptamer, human luteinizing hormone, human chorionic gonadotropin, degrelig acetate (antagonist of LHRH receptor) or StemRegenin 1 as targeting ligands.

[0174] In certain embodiments, the targeting ligand that binds to CD34 is a human or humanized antibody. In certain embodiments, the targeting ligand that binds to CD34 is antibody clone: ​​581; antibody clone: ​​561; antibody clone: ​​REA1164; or antibody clone: ​​AC136; or a binding fragment derived therefrom.

[0175] In a specific embodiment, the binding domain that binds to CD34 comprises a variable light chain comprising a CDRL1 sequence comprising RSSQTIVHSNGNTYLE (SEQ ID NO: 139), a CDRL2 sequence comprising QVSNRFS (SEQ ID NO: 140), a CDRL3 sequence comprising FQGSHVPRT (SEQ ID NO: 141), a CDRH1 sequence comprising GYTFTNYGMN (SEQ ID NO: 142), a CDRH2 sequence comprising WINTNTGEPKYAEEFKG (SEQ ID NO: 143), and a CDRH3 sequence comprising GYGNYARGAWLAY (SEQ ID NO: 144). For more information on binding domains that bind to CD34, see WO2008CN01963. Other CD34 binding domains are also commercially available. For example, Invitrogen provides a CD34 monoclonal antibody (QBEND / 10; clone: ​​QBEnd / 10; catalog number: MA1-10202).

[0176] In specific embodiments, the binding domain that binds CD90 is antibody clone: ​​5E10; antibody clone: ​​DG3; antibody clone: ​​REA897; or a binding fragment derived therefrom.

[0177] In a specific embodiment, the binding domain that binds to CD90 is a single chain antibody comprising the sequence CMASASQVQLVQSGAEVKKPGASVKVSCKASGYTFTG YYVHWVRQAPGQGLEWMGWVNPNSGDTNYAQKFQGRVTMTRDTSISTAYMELSGLRSDDTAVYYCARDGDEDWYFDLWGRGTPVTVSSGILGSGGGGSGGGGSGGGGSDIRLTQSPSSLSASIGDRVTITCRASQGISRSLVWYQQKPGKAPRLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHNTYPFTFGPGTK VDIKSGIPEQKL (SEQ ID NO: 145). In a specific embodiment, the binding domain is human or humanized. For more information about binding domains that bind to CD90, see WO2017U S35989. CD90 binding domains are also commercially available. For example, Abcam provides anti-CD90 / Thy1 antibody ([EPR3133]; clone: ​​EPR3133; catalog number: ab133350).

[0178] In specific embodiments, the binding domain that binds to CD133 is antibody clone: ​​REA820; antibody clone: ​​REA753; antibody clone: ​​REA816; antibody clone: ​​293C3; antibody clone: ​​AC141; antibody clone: ​​AC133; antibody clone: ​​7; or a binding fragment derived therefrom.

[0179] In a specific embodiment, the binding domain that binds to CD133 is derived from C178A BC-CD133 MAb. In a specific embodiment, the binding domain comprises a variable light chain NI VMTQSPKSMSMSLGERVTLSCKASENVDTYVSWYQQKPEQSPK VLIYGASNRYTGVPDRFTGSGSATDFSLTISNVQAEDLADYHCGQ SYRYPLTFGAGTKLELKR (SEQ ID NO: 146) and a variable heavy chain EIQLQQS GPDLMKPGASVKISCKASGYSFTNYYVHWVKQSLDKSLEWIGYV DPFNGDFNYNQKFKDKATLTVDKSSSTAYMHLSSLTSEDSAVYY CARGGLDWYDTSYWYFDVWGAGTAV (SEQ ID NO: 147).

[0180] In a specific embodiment, the binding domain comprises a variable light chain comprising a CDRL1 sequence comprising QSSQSVYNNNYLA (SEQ ID NO: 148), a CDRL2 sequence comprising RASTLAS (SEQ ID NO: 149), a CDRL3 sequence comprising QGEFSCDSADCAA (SEQ ID NO: 150), a CDRH1 sequence comprising GIDLNNY (SEQ ID NO: 151), a CDRH2 sequence comprising FGSDS (SEQ ID NO: 152), and a CDRH3 sequence comprising GGL.

[0181] In a specific embodiment, the binding domain is human or humanized. For more information about binding domains for CD133, see WO2011089211, U.S. Publication No. 2018 / 0105598 and / or U.S. Publication No. 2013 / 0224202. CD133 binding domains are also commercially available. For example, Abcam provides anti-CD133 antibodies ([EPR20980-45; Clone: ​​EPR20980-45; Catalog No.: ab226355).

[0182] In a specific embodiment, the binding domain that binds to CD133 is an aptamer. The aptamer can be aptamer A15 or B19 from Tocris Biosciences. In a specific embodiment, aptamer A15 refers to a 15-base and formula C 182 H 219 F9N 58 O 104 P 16 RNA aptamer. The molecular weight of the tissue aptamer is 5549.58, and the sequence is modified as follows: 2-fluoropyrimidine, 3'-inverted deoxythymidine cap, 5'-fluorescent DY647 tag. See also Shigdar et al. (2013) RNA aptamers targeting cancer stem cell marker CD133. Cancer Lett. 330 84 PMID: 23196060. In a specific embodiment, aptamer B19 refers to an RNA aptamer having 19 bases and the formula C221H263F10N73O131P20. The tissue aptamer has a molecular weight of 6847.32, and the sequence is modified as follows: 2-fluoropyrimidine, 3'-inverted deoxythymidine cap, 5'-fluorescent DY647 tag. See also Shigdar et al. (2013) RNA aptamers targeting cancer stem cell marker CD133. Cancer Lett. 330 84 PMID: 23196060

[0183] In a specific embodiment, the RNA aptamer comprises a consensus sequence comprising CCCUCCUACAUAG GG (SEQ ID NO: 153). In a specific embodiment, the RNA aptamer comprises a consensus sequence comprising GAGACAAGAAUAAACGCUC AACCCACCCUCCUACAUAGGGAGGAACGAGUUACUAUAGA GCUUCGACAGGAGGCUCACAAC (SEQ ID NO: 154);

[0184] GAGACAAGAAUAAACGCUCAACCCACCCUCCUACAUAGG GAGGAACGAGUUACUAUAG(SEQ IDNO:155);

[0185] GCUCAACCCACCCUCCUACAUAGGGAGGAACGAGU (SEQ ID NO: 111);

[0186] CCACCCUCCUACAUAGGGUGG (SEQ ID NO: 156); CAGA ACGUAUACUAUUCUG (SEQ ID NO: 157);

[0187] AGAACGUAUACUAUU (SEQ ID NO: 158); or

[0188] GAGACAAGAAUAAACGCUCAAGGAAAGCGCUUAUUGUU GCCUAUGUUAGAACGUAUACUAUUUCGACAGGAGGCUCAC AACAGGC (SEQ ID NO: 159). For additional information on CD133 aptamers, see EP2880185.

[0189] Specific embodiments use a targeting ligand that binds to the luteinizing hormone receptor (LHR). Specific embodiments can utilize LH alpha subunits and LH beta subunits. In specific embodiments, the alpha subunit comprises DCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPL RSKKTMLVQKNVTSESTCCVAKSYNRV TVMGGFKVENHTACH CSTCYYHKS (human) (SEQ ID NO: 53) or GCPECKLKENKYFSKLGA PIYQCMGCCFSRAYPTPARSKKTMLVPKNITSEATCCVAKAFTKA TVMGNARVENHTECHCSTCYYHKS (mouse) (SEQ ID NO: 54).

[0190] In specific embodiments, the LHβ subunit comprises SREPLRPWCHPINAILA VEKEGCPVCITVNTTICAGYCPTMMRVLQAVLPPLPQVVCTYRD VRFESIRLPGCPRGVDPVVSFPVALSCRCGPCRRSTSDCGGPKDHPLTCDHPQLSGLLFL (human) (SEQ ID NO: 55) or SRGPLRPLCRPVN ATLAAENEFCPVCITFTTSICAGYCPSMVRVLPAALPPVPQPVCTYRELRFASVRLPGCPPGVDPIVSFPVALSCRCGPCRLSSSDCGGPRTQPMACDLPHLPGLLLL (mouse) (SEQ ID NO: 56).

[0191] Many antibodies that bind to LHR or other HSC1 / HSC2 markers are commercially available. For example, anti-LHR antibodies can be purchased from Abcam, Invitrogen, Alomone Labs, Novus Biologicals, Origene Technologies, Bio-Rad, Abbexa, St. John's Laboratory, Millipore Sigma (Burlington, MA), LifeSpan Biosciences, etc.

[0192] In a specific embodiment, an anti-LHR binder comprises a CDRH1 comprising GYSITSGYG (SEQ ID NO:57); a CDRH2 comprising IHYSGST (SEQ ID NO:58); a CDRH3 comprising ARSLRY (SEQ ID NO:59); and a CDRL1 comprising SSVNY (SEQ ID NO:60); a CDRL2 comprising DTS; and a CDRL3 comprising HQWSSYPYT (SEQ ID NO:61).

[0193] In a specific embodiment, the anti-LHR binder comprises a CDRH1 comprising GFSLTTYG (SEQ ID NO:62); a CDRH2 comprising IWGDGST (SEQ ID NO:63); and a CDRH3 comprising AEGSSLFAY (SEQ ID NO:64); and a CDRL1 comprising QSLLNSGNQKNY (SEQ ID NO:65); a CDRL2 comprising WAS; and a CDRL3 comprising QNDYSYPLT (SEQ ID NO:66).

[0194] In a specific embodiment, the anti-LHR binder comprises a CDRH1 comprising GYSFTGYY (SEQ ID NO:67); a CDRH2 comprising IYPYNGVS (SEQ ID NO:68); and a CDRH3 comprising ARERGLYQLRAMDY (SEQ ID NO:69); and a CDRL1 comprising QSISNN (SEQ ID NO:70); a CDRL2 comprising NAS; and a CDRL3 comprising QQSNSWPYT (SEQ ID NO:71).

[0195] In a specific embodiment, an anti-LHR binding agent comprises a heavy chain comprising EVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYGWHRQFP GNKLEWMGYIHYSGSTTYNPSLKSRISISRDTSKNQFFLQLNSVTTEDTATYYCARSLRYWGQGTTLTVSS (SEQ ID NO: 72) and a light chain comprising DIVMTQTPAIMSASPGQKVTITCSASSSVNYMHWYQQKLGSPKLWIYDTSKLAPGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPYTFGSGTKLEIK (SEQ ID NO: 73).

[0196] In a specific embodiment, an anti-LHR binding agent comprises a heavy chain comprising QVQLKESGPGLVAPSQSLSrrCTVSGFSLTTYGVSWVRQPP GKGLEWLGVIWGDGSTYYHSALISRLSISKDNSKSQVFLKLNSLQTDDTATYYCAEGSSLFAYWGQGTLVTVSA (SEQ ID NO: 74) and a light chain comprising DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRQSGVPDRFTGSGSGTDFTLTISSVQAEDXAVYYCQNDYSYPLTFGSGTKLEIK (SEQ ID NO: 75).

[0197] In a specific embodiment, an anti-LHR binding agent comprises a heavy chain comprising EVQLEQSGGGLVQPGGSRKLSCAASGFTFSSFGMHWVRQ APEKGLEWVAYISSGSSTLHYADTVKGRFTISRDNPKNTLFLQMKLPSLCYGLLGSRNLSHRLL (SEQ ID NO:76) and a light chain comprising DIV LTQTPSSLSASLGDTITITCHASQNINVWLFWYQQKPGNIPKLLIY KASNLLTGVPSRFSGSGSGTGFTLTISSLQPEDIATYYCQQGQSFPWTFGGGTKLEIK (SEQ ID NO:77).

[0198] In a specific embodiment, an anti-LHR binding agent comprises a heavy chain comprising QVKLQQSGPELVKPGASVKISCKASGYSFTGYYMHWVKQ SHGNILDWIGYIYPYNGVSSYNQKFKGKATLTVDKSSSTAYMELR SLTSEDSAVYYCARERGLYQLRAMDYWGQGTSVTVSS (SEQ ID NO: 78) and a light chain comprising DIVLTQTPATLSVTPGDSVSLSCRASQSIS NNLHWYQQKSHESPRLLIKNASQSISGIPSKFSGSGSGTDFTLRINS VETEDFGMYFCQQSNSWPYTFGSGTKLEIK (SEQ ID NO: 79).

[0199] In a specific embodiment, the anti-LHR binding agent comprises subunit β3 of human chorionic gonadotropin (CGB3; UniProt ID P0DN86) comprising SKEPLRPRCRPINATLAVEKEGCPVCITVNTTICAGYCPTMTRVLQGVLPALPQWCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCALCRRSTTDCGG PKDHPLTCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 160).

[0200] Specific embodiments include the use of targeting ligands that bind to the aryl hydrocarbon receptor (AHR). AHR is a member of the basic helix-loop-helix transcription factor family. AHR regulates the function of xenobiotic metabolizing enzymes and the toxicity and carcinogenic properties of several compounds. AHR also plays an important role in the regulation of the pluripotency and stemness of HSCs. It has been shown that inhibition of AHR by StemRegenin 1 (SR1) leads to an increase in cells expressing CD34 and an increase in cells that retain the ability to be implanted into immunodeficient mice.

[0201] In certain embodiments, SR1, also known as 4-(2-((2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-yl)amino)ethyl)phenol, has the formula C 24 H 23 N5OS and the following structure:

[0202]

[0203] SR1 is available from sources such as Cayman Chemical Company, Ann Arbor, MI; STEMCELL TM Technologies, Vancouver, CA; and Abcam, Cambridge, MA.

[0204] In a specific embodiment, the binding domain of the selected cell targeting ligand comprises a T-cell receptor motif antibody; a T-cell alpha chain antibody; a T-cell beta chain antibody; a T-cell gamma chain antibody; a T-cell delta chain antibody; a CCR7 antibody; a CD1a antibody; a CD1b antibody; a CD1c antibody; a CD1d antibody; a CD3 antibody; a CD4 antibody; a CD5 antibody; a CD7 antibody; a CD8 antibody; a CD11b antibody; a CD11c antibody; a CD16 antibody; a CD19 antibody; a CD20 antibody; a CD21 antibody; a CD22 antibody; a CD25 antibody; a CD28 antibody; a CD34 antibody; a CD35 antibody; a CD39 antibody; a CD4 0 antibody; CD45RA antibody; CD45RA antibody; CD46 antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD86 antibody; CD90 antibody; CD95 antibody; CD101 antibody; CD117 antibody; CD127 antibody; CD137 (4-1BB) antibody; CD148 antibody; CD163 antibody; CD164 antibody; F4 / 80 antibody; IL-4Rα antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody; GARP antibody; LAP antibody; granzyme B antibody; LFA-1 antibody; or transferrin receptor antibody.

[0205] Targeting ligands that enable selective delivery of NPs to T cells can include a binding domain that binds CD3 derived from at least one of OKT3 (described in U.S. Pat. No. 5,929,212), otelezumab, teluzumab, visilizumab, 20G6-F3, 4B4-D7, 4E7-C9, 18F5-H10, or TR66. In specific embodiments, the binding domain comprises the variable light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVY YCQQRSNWPPLTFGGGTKVEIK (SEQ ID NO: 161) and the variable heavy chain Q VQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKG LEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQMGYWHFDLWGRGTLVTVSS (SEQ ID NO: 162).

[0206] In a specific embodiment, the binding domain comprises the variable light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK (SEQ ID NO: 161) and the variable heavy chain QVQLVQSGGGWQSGRSLRLSCAASGFKFSGYGMHWVRQAPGKG LEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRGEDTAVYYCARQMGYWHFDLWGRGTLVTVSS (SEQ ID NO: 163).

[0207] In a specific embodiment, the binding domain comprises a variable light chain comprising a CDRL1 sequence comprising SASSVSYMN (SEQ ID NO: 164), a CDRL2 sequence comprising RWIYDTSKLAS (SEQ ID NO: 165), a CDRL3 sequence comprising QQWSSNPFT (SEQ ID NO: 166), a CDRH1 sequence comprising KASGYTFTRYTMH (SEQ ID NO: 167), a CDRH2 sequence comprising INPSRGYTNYNQKFKD (SEQ ID NO: 168), and a CDRH3 sequence comprising YYDDHYCLDY (SEQ ID NO: 169).

[0208] In a specific embodiment, the binding domain comprises a variable light chain comprising a CDRL1 sequence comprising QSLVHNNGNTY (SEQ ID NO: 170), a CDRL2 sequence comprising KVS, a CDRL3 sequence comprising GQGTQYPFT (SEQ ID NO: 171), a CDRH1 sequence comprising GFTFTKAW (SEQ ID NO: 172), a CDRH2 sequence comprising IKDKSNSYAT (SEQ ID NO: 173), and a CDRH3 sequence comprising RGVYYALSPFDY (SEQ ID NO: 174).

[0209] In a specific embodiment, the binding domain comprises a variable light chain comprising a CDRL1 sequence comprising QSLVHDNGNTY (SEQ ID NO: 175), a CDRL2 sequence comprising KVS, a CDRL3 sequence comprising GQGTQYPFT (SEQ ID NO: 171), a CDRH1 sequence comprising GFTFSNAW (SEQ ID NO: 175), a CDRH2 sequence comprising IKARSNNYAT (SEQ ID NO: 176), and a CDRH3 sequence comprising RGTYYASKPFDY (SEQ ID NO: 177).

[0210] In a specific embodiment, the binding domain comprises a variable light chain comprising a CDRL1 sequence comprising QSLEHNNGNTY (SEQ ID NO: 179), a CDRL2 sequence comprising KVS, a CDRL3 sequence comprising GQGTQYPFT (SEQ ID NO: 171), a CDRH1 sequence comprising GFTFSNAW (SEQ ID NO: 176), a CDRH2 sequence comprising IKDKSNNYAT (SEQ ID NO: 180), and a CDRH3 sequence comprising RYVHYGIGYAMDA (SEQ ID NO: 181).

[0211] In a specific embodiment, the binding domain comprises a variable light chain comprising a CDRL1 sequence comprising QSLVHTNGNTY (SEQ ID NO: 182), a CDRL2 sequence comprising KVS, a CDRL3 sequence comprising GQGTHYPFT (SEQ ID NO: 183), a CDRH1 sequence comprising GFTFTNAW (SEQ ID NO: 184), a CDRH2 sequence comprising KDKSNNYAT (SEQ ID NO: 185), and a CDRH3 sequence comprising RYVHYRFAYALDA (SEQ ID NO: 186).

[0212] In certain embodiments, the binding domain is human or humanized. For more information on binding domains that bind to CD3, see U.S. Pat. No. 8,785,604, PCT / US 17 / 42264, and / or WO02051871. CD3 binding domains are also commercially available. For example, LSBio offers PathPlus TM CD3 antibody monoclonal IHC LS-B8669 (clone: ​​SP7; catalog number: LS-B8669-100).

[0213] T cells expressing CD4 can be targeted for selective NP delivery, wherein the binding domain that binds CD4 is an antibody. In a specific embodiment, the binding domain comprises a variable light chain DIVMTQSPDSLAVSLGERVTMNCKSSQSLLYSTNQKNYLAWYQQ KPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVA VYYCQQYYSYRTFGGGTKLEIK (SEQ ID NO: 187) and a variable heavy chain QVQLQQSGPEVVKPGASVKMSCKASGYTFTSYVIHWVRQKPGQ GLDWIGYINPYNDGTDYDEKFKGKATLTSDTSTSTAYMELSSLRS EDTAVYYCAREKDNYATGAWFAYWGQGTLVTVSS (SEQ ID NO: 188). In a specific embodiment, the binding domain comprises a variable light chain comprising a CDRL1 sequence comprising KSSQSLLYSTNQKNYLA (SEQ ID NO: 189), a CDRL2 sequence comprising WASTRES (SEQ ID NO: 190), a CDRL3 sequence comprising QQYYSYRT (SEQ ID NO: 191), a CDRH1 sequence comprising GYTFTSYVIH (SEQ ID NO: 192), a CDRH2 sequence comprising YINPYNDGTDYDEKFKG (SEQ ID NO: 193), and a CDRH3 sequence comprising EKDNYATGAWFAY (SEQ ID NO: 194). In a specific embodiment, the binding domain is human or humanized. For more information about binding domains that bind to CD4, see PCT Application No. WO2008US05450. CD4 binding domains are also commercially available. For example, R&D Systems provides human CD4 antibodies (clone: ​​34930; catalog number: MAB379).

[0214] CD28 is a surface glycoprotein present on 80% of human peripheral T cells and is present on resting and activated T cells. CD28 binds to B7-1 (CD80) and B7-2 (CD86). In a specific embodiment, the CD28 binding domain (e.g., scFv) is derived from CD80, CD86 or 9D7 antibodies. Other antibodies that bind to CD28 include 9.3, KOLT-2, 15E8, 248.23.2 and EX5.3D10. In addition, 1YJD provides a crystal structure of human CD28 complexed with the Fab fragment of a mitogenic antibody (5.11A1). In a specific embodiment, an antibody that does not compete with 9D7 is selected.

[0215] In a specific embodiment, the CD28 binding domain is derived from TGN1412. In a specific embodiment, the variable heavy chain of TGN1412 comprises: QVQLVQSGAEVKKP GASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGL DWNFDVWGQGTTVTVSS (SEQ ID NO: 195), and the variable light chain of TGN1412 comprises: DIQMTQSPSSLSASVGDRVTITCHASQNIYVWLNW YQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQGQTYPYTFGGGTKVEIK (SEQ ID NO: 196).

[0216] In a specific embodiment, the CD28 binding domain comprises a variable light chain and a variable heavy chain, wherein the variable light chain comprises a CDRL1 sequence comprising HASQNIYVWLN (SEQ ID NO: 197), a CDRL2 sequence comprising KASNLHT (SEQ ID NO: 198), and a CDRL3 sequence comprising QQGQTYPYT (SEQ ID NO: 199), and the variable heavy chain comprises a CDRH1 sequence comprising GYTFTSYYIH (SEQ ID NO: 200), a CDRH2 sequence comprising CIYPGNVNTNYNEK (SEQ ID NO: 201), and a CDRH3 sequence comprising SHYGLDWNFDV (SEQ ID NO: 202).

[0217] In a specific embodiment, the CD28 binding domain comprises a variable light chain and a variable heavy chain, wherein the variable light chain comprises a CDRL1 sequence comprising HASQNIYVWLN (SEQ ID NO: 197), a CDRL2 sequence comprising KASNLHT (SEQ ID NO: 198), and a CDRL3 sequence comprising QQGQTYPYT (SEQ ID NO: 199), and the variable heavy chain comprises a CDRH1 sequence comprising SYYIH (SEQ ID NO: 203), a CDRH2 sequence comprising CIYPGNVNTNYNEKFKD (SEQ ID NO: 204), and a CDRH3 sequence comprising SHYGLDWNFDV: (SEQ ID NO: 202).

[0218] Activated T cells express 4-1BB (CD137). In a specific embodiment, the 4-1BB binding domain comprises a variable light chain and a variable heavy chain, wherein the variable light chain comprises a CDRL1 sequence containing RASQSVS (SEQ ID NO: 205), a CDRL2 sequence containing ASNRAT (SEQ ID NO: 206), and a CDRL3 sequence containing QRSNWPPALT (SEQ ID NO: 207), and the variable heavy chain comprises a CDRH1 sequence containing YYWS (SEQ ID NO: 208), a CDRH2 sequence containing INH, and a CDRH3 sequence containing YGPGNYDWYFDL (SEQ ID NO: 209).

[0219] In a specific embodiment, the 4-1BB binding domain comprises a variable light chain and a variable heavy chain, wherein the variable light chain comprises a CDRL1 sequence comprising SGDNIGDQYAH (SEQ ID NO: 210), a CDRL2 sequence comprising QDKNRPS (SEQ ID NO: 211), and a CDRL3 sequence comprising ATYTGFGSLAV (SEQ ID NO: 212), and the variable heavy chain comprises a CDRH1 sequence comprising GYSFSTYWIS (SEQ ID NO: 213), a CDRH2 sequence comprising KIYPGDSYTNYSPS (SEQ ID NO: 101), and a CDRH3 sequence comprising GYGIFDY (SEQ ID NO: 102).

[0220] Specific embodiments disclosed herein include targeting ligands that bind to epitopes on CD8. In specific embodiments, a CD8 binding domain (e.g., scFv) is derived from an OKT8 antibody. For example, in specific embodiments, a CD8 binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable light chain, the variable light chain comprising a CDRL1 sequence containing RTSRSISQYLA (SEQ ID NO: 103), a CDRL2 sequence containing SGSTLQS (SEQ ID NO: 104), and a CDRL3 sequence containing QQHNENPLT (SEQ ID NO: 105). In specific embodiments, a CD8 binding domain is a human or humanized binding domain (e.g., scFv) comprising a variable heavy chain, the variable heavy chain comprising a CDRH1 sequence containing GFNIKD (SEQ ID NO: 106), a CDRH2 sequence containing RIDPANDNT (SEQ ID NO: 107), and a CDRH3 sequence containing GYGYYVFDH (SEQID NO: 108). These reflect the CDR sequences of the OKT8 antibody.

[0221] Examples of commercially available antibodies having a binding domain that binds to NK cell receptors include: 5C6 and 1D11 (available from San Diego, CA); mAb33 binding to KIR2DL4 (available from ); P44-8 that binds to NKp44 (available from ), SK1 that binds CD8, and 3G8 that binds CD16. The binding domains that bind KIR2DL1 and KIR2DL2 / 3 comprise a variable light chain region of the sequence EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSN WMYTFGQGTKLEIKRT (SEQ ID NO: 109) and a variable heavy chain region of the sequence QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQG LEWMGGFIPIFGAANYAQKFQGRVTITADESTSTAYMELSSLRSD DTAVYYCARIPSGSYYYDYDMDVWGQGTTVTVSS (SEQ ID NO: 110). Other NK binding antibodies are described in WO / 2005 / 0003172 and US Pat. No. 9,415,104.

[0222] Commercially available antibodies that bind to proteins expressed on the surface of macrophages include M1 / 70 (available from BioLegend) that binds to CD11b; KP1 (available from ABCAM, Cambridge, United Kingdom) that binds to CD68; and ab87099 (available from ABCAM) that binds to CD163.

[0223] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described in Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al. (1997) J Mol Biol 273:927-948 (Chothia numbering scheme); Maccallum et al. (1996) J Mol Biol 262:732-745 (Contact numbering scheme); Martin et al. (1989) Proc. Natl. Acad. Sci., 86:9268-9272 (AbM numbering scheme; Lefranc MP et al. (2003) Dev Comp Immunol 27(1):55-77 (IMGT numbering scheme); and Honegger and Pluckthun (2001) J Mol Biol 309(3):657-670 ("Aho" numbering scheme). The boundaries of a given CDR or FR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering of the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, with insertions accommodated by inserting letters such as "30a", and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on analysis of composite crystal structures and is similar to the Chothia numbering scheme in many respects. In a specific embodiment, the antibody CDR sequences disclosed herein are numbered according to Kabat.

[0224] In a specific embodiment, when it is desired to obtain functional genetic modification, selective delivery can be enhanced by including regulatory elements that restrict the expression of the inserted construct to the desired / selected cell type. For example, for HSC, CD45 promoter, Wiskott-Aldrich syndrome (WASP) promoter or interferon (IFN)-β promoter can be used; for HSC or T cells, mouse stem cell virus promoter or distal lck promoter can be used; or for B cells, selective delivery can be enhanced by using B29 promoter.

[0225] Other agents that may also promote lymphocyte internalization and / or transfection, such as poly(ethyleneimine) / DNA (PEI / DNA) complexes, may also be used.

[0226] In certain embodiments, the targeting ligand can be linked to the nuclease using, for example, an amine-thiol or thiol-thiol crosslinker with various PEG spacers and / or Gly-Ser spacers. The addition of spacers allows flexibility in binding to cognate receptors or cell surface proteins. In certain embodiments, the spacer can have between 1-50; 10-50; 20-50; 30-50; 1-500; 10-250; 20-200; 30-150; 40-100; 50-75; or 5-75 repeating units or residues.

[0227] (V) Sources and processing of cell populations. Sources of HSC, HSPC and other lymphocytes include cord blood, placental blood, bone marrow, peripheral blood, embryonic cells, aorta-gonad-mesonephros derived cells, lymph, liver, thymus and spleen from age appropriate donors. Methods for the collection and processing of biological samples including blood samples are known. See, e.g., Alsever et al., 1941, NY St. J. Med. 41:126; De Gowin, et al., 1940, J. Am. Med. Ass. 114:850; Smith, et al., 1959, J. Thorac. Cardiovasc. Surg. 38:573; Rous and Turner, 1916, J. Exp. Med. 23:219; and Hum, 1968, Storage of Blood, Academic Press, New York, pp. 26-160; Kodo et al., 1984, J. Clin Invest. 73:1377-1384), all collected samples can be screened for unwanted components and discarded, processed or used according to current standards recognized at the time. In a specific embodiment, the biological sample includes any biological fluid, tissue, blood cell product and / or organ containing a target cell population.

[0228] Any procedure generally known in the art can be used to obtain a source or biological sample including a target cell colony from a subject. In a specific embodiment, the HSC / HSPC in the peripheral blood is mobilized before collection. Peripheral blood HSC / HSPC can be mobilized by any method. Peripheral blood HSC / HSPC can be mobilized by treating the subject with any medicaments described herein or known in the art that increase the number of HSC / HSPC circulating in the peripheral blood of the subject. For example, in a specific embodiment, peripheral blood is mobilized by treating the subject with one or more cytokines or growth factors (e.g., G-CSF, kit ligand (KL), IL-1, IL-7, IL-8, IL-11, Flt3 ligand, SCF, thrombopoietin or GM-CSF (such as sargramostim)). Different types of G-CSF that can be used for peripheral blood mobilization methods include filgrastim and long-acting G-CSF-polyethylene glycol filgrastim. In certain embodiments, the chemokine receptor ligands (e.g., chemokine receptor 2 ligands GROβ and GROβ) are used to target the chemokine receptor 2 ligands. Δ4 ), chemokine receptor analogs (e.g., stromal cell derived factor-1α (SDF-1a) protein analogs, such as CTCE-0021, CTCE-0214 or SDF-1α, such as Met-SDF-1β) or chemokine receptor antagonists (e.g., chemokine (CXC motif) receptor 4 (CXCR4) antagonists, such as AMD3100) to mobilize peripheral blood.

[0229] In specific embodiments, peripheral blood is mobilized by treating the subject with one or more anti-integrin signaling agents (eg, function-blocking anti-very late antigen 4 (VLA-4) antibodies or anti-vascular cell adhesion molecule 1 (VCAM-1)).

[0230] Peripheral blood can be mobilized by treating the subject with one or more cytotoxic drugs such as cyclophosphamide, etoposide, or paclitaxel.

[0231] In a specific embodiment, peripheral blood can be mobilized by applying one or more of the above-mentioned agents to the subject for a period of time. For example, before collecting HSC / HSPC, one or more agents (for example, G-CSF) can be used to treat the subject by injection (for example, subcutaneous, intravenous or intraperitoneal), once a day or twice a day, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days. In a specific embodiment, HSC / HSPC is collected in 1, 2, 3, 4, 5, 6, 7, 8, 12, 14, 16, 18, 20 or 24 hours after the last dose of medicament for mobilizing HSC / HSPC into peripheral blood. In certain embodiments, HSC / HSPC are mobilized by treating the subject with two or more different types of agents described above or known in the art, such as a growth factor (e.g., G-CSF) and a chemokine receptor antagonist (e.g., a CXCR4 receptor antagonist such as AMD3100) or a growth factor (e.g., G-CSF or KL) and an anti-integrin agent (e.g., a function-blocking VLA-4 antibody). The different types of mobilizing agents may be administered simultaneously or sequentially. For additional information on peripheral blood mobilization methods, see, e.g., Craddock et al., 1997, Blood 90(12):4779-4788; Jin et al., 2008, Journal of Translational Medicine 6:39; Pelus, 2008, Curr. Opin. Hematol. 15(4):285-292; Papayannopoulou et al., 1998, Blood 91(7):2231-2239; Tricot et al., 2008, Haematologica 93(11):1739-1742; and Weaver et al., 2001, Bone Marrow Transplantation 27(2):S23-S29).

[0232] HSC / HSPC from peripheral blood can be collected from the blood by a syringe or catheter inserted into the subject's vein. For example, in a specific embodiment, a single collection machine can be used to collect peripheral blood. The blood flows from the vein through a catheter into the single collection machine, which separates the white blood cells including HSC / HSPC from the remaining blood and then returns the remaining blood to the subject. Single collection can be performed for several days (e.g., 1 to 5 days) until enough selected cell types (e.g., HSC, T cells) are collected.

[0233] In certain embodiments, no further collection or separation of the selected cell type is required before exposing the obtained sample to the NPs disclosed herein, as the NPs selectively target the selected cell type within the heterogeneous cell population. In certain embodiments, no other manipulation is performed on the obtained sample except for the NP addition.

[0234] In some embodiments, the blood cells collected from the subject are washed, for example to remove the plasma portion and the cells are placed in a suitable buffer or culture medium for subsequent exposure to NP. In a specific embodiment, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium and / or magnesium and / or many or all divalent cations. Washing can be completed using a semi-automatic "flow-through" centrifuge (e.g., Cobe2991 cell processor, Baxter) according to the manufacturer's instructions. Tangential flow filtration (TFF) can also be performed. In a specific embodiment, the cells can be resuspended in various biocompatible buffers after washing, such as PBS without Ca++ / Mg++.

[0235] In certain embodiments, some limited further cell collection and separation may be beneficial prior to exposure to the NPs disclosed herein. In certain embodiments, any suitable technique may be used to collect and separate selected cell types from a sample. Suitable collection and separation procedures include magnetic separation; fluorescence activated cell sorting (FACS; Williams et al., 1985, J. Immunol. 135: 1004; Lu et al., 1986, Blood 68 (1): 126-133); affinity chromatography; agents combined with or used in conjunction with monoclonal antibodies; "panning" with antibodies attached to a solid matrix (Broxmeyer et al., 1984, J. Clin. Invest. 73: 939-953); selective agglutination using lectins (such as soybeans) (Reisner et al., 1980, Proc. Natl. Acad. Sci. USA 77: 1164), etc. Certain embodiments may utilize limited separation. Limited separation refers to crude cell enrichment, for example by removal of erythrocytes and / or adherent phagocytes.

[0236] In a particular embodiment, CD34+ HSPCs can be used, for example, using antibodies directly or indirectly conjugated to magnetic particles in conjunction with a magnetic cell separator, e.g. The cell separation system (Miltenyi Biotec, Bergisch Gladbach, Germany) processes the subject sample (e.g., blood sample) to select / enrich the Figure 2In certain embodiments where some limited cell enrichment is performed, cells within a sample may be enriched based on CD34 alone; CD133+ alone; CD90+ alone; CD164+ alone; CD46+ alone; or LH+ alone. In certain embodiments, cells may be enriched and / or isolated based on one or more of various combinations of CD34; CD133+; CD90+; CD164+; CD46+; AHR+; or LH+. In certain embodiments, LH+ means that the cells express the LHRH receptor. In certain embodiments, AHR+ means that the cells express the aryl hydrocarbon receptor.

[0237] When implementing reduced but not minimal manufacturing, expansion of HSC / HSPC may be useful. Expansion can occur in the presence of one or more growth factors, such as: angiopoietin-like protein (Angptl, e.g., Angptl2, Angptl3, Angptl7, Angpt15, and Mfap4); erythropoietin; fibroblast growth factor-1 (FGF-1); Flt-3 ligand (Flt-3L); granulocyte colony stimulating factor (G-CSF); granulocyte-macrophage colony stimulating factor (GM-CSF); insulin growth factor-2 (IFG -2); interleukin-3 (IL-3); interleukin-6 (IL-6); interleukin-7 (IL-7); interleukin-11 (IL-11); stem cell factor (SCF; also known as c-kit ligand or mast cell growth factor); thrombopoietin (TPO); and their analogs (wherein the analogs include any structural variants of growth factors that have the biological activity of naturally occurring growth factors; see, e.g., WO2007 / 1145227 and U.S. Patent Publication No. 2010 / 0183564).

[0238] In certain embodiments, the amount or concentration of growth factor suitable for expanding HSC / HSPC or lymphocytes is an amount or concentration effective to promote proliferation. The lymphocyte population is preferably expanded until a sufficient number of cells is obtained to provide at least one infusion into a human subject, typically about 10 4 cells / kg to 10 9 cells / kg.

[0239] The amount or concentration of growth factors suitable for expanding HSC / HSPC or lymphocytes depends on the activity of the growth factor preparation, the species correspondence between the growth factor and the lymphocyte, etc. Typically, when the growth factor and the lymphocyte belong to the same species, the total amount of growth factor in the culture medium is in the range of 1 ng / ml to 5 μg / ml, 5 ng / ml to 1 μg / ml, or 5 ng / ml to 250 ng / ml. In specific embodiments, the amount of growth factor can be in the range of 5-1000 or 50-100 ng / ml.

[0240] In a specific embodiment, the growth factors are present in the expansion culture conditions at the following concentrations: 25-300 ng / ml SCF, 25-300 ng / ml Flt-3L, 25-100 ng / ml TPO, 25-100 ng / ml IL-6, and 10 ng / ml IL-3. In a specific embodiment, 50, 100, or 200 ng / ml SCF; 50, 100, or 200 ng / ml Flt-3L; 50 or 100 ng / ml TPO; 50 or 100 ng / ml IL-6; and 10 ng / ml IL-3 may be used.

[0241] HSC / HSPC or lymphocytes can be expanded in tissue culture dishes on which extracellular matrix proteins such as fibronectin (FN) or fragments thereof (e.g., CH-296 (Dao et al., 1998, Blood 92(12):4612-21)) or (Recombinant human fibronectin fragment; (Clontech Laboratories, Inc., Madison, Wl).

[0242] Notch agonists are particularly useful for expanding HSC / HSPC. In specific embodiments, HSC / HSPC can be expanded by exposing HSC / HSPC to immobilized Notch agonists and 50ng / ml or 100ng / ml SCF; immobilized Notch agonists and 50ng / ml or 100ng / ml Flt-3L, IL-6, TPO, and SCF, respectively; or immobilized Notch agonists and 50ng / ml or 100ng / ml Flt-3L, IL-6, TPO, and SCF, and 10ng / ml IL-11 or IL-3.

[0243] For additional general information on suitable culture and / or expansion conditions, see U.S. Pat. No. 7,399,633; U.S. Patent Publication No. 2010 / 0183564; Freshney Culture of Animal Cells, Wiley-Liss, Inc., New York, NY (1994); Vamum-Finney et al., 1993, Blood 101:1784-1789; Ohishi et al., 2002, J. Clin. Invest. 110:1165-1174; Delaney et al., 2010, Nature Med. 16(2):232-236; WO 2006 / 047569 A2; WO 2007 / 095594 A2; U.S. Pat. No. 5,004,681; WO 2011 / 127470 A1; WO 2011 / 127472 A1; and see Regenerative Medicine 2011. Medicine, Department of Health and Human Services Chapter 2, August 2006, and references cited therein.

[0244] When reduced, but not minimal, manipulations are performed, samples can be enriched for T cells by using density-based cell separation methods and related methods. For example, leukocytes can be separated from other cell types in peripheral blood by lysing red blood cells and centrifuging the sample through a Percoll or Ficoll gradient.

[0245] In a specific embodiment, a large number of T cell populations that are not enriched for a specific T cell type can be used. In a specific embodiment, the selected T cell type can be enriched and / or separated according to positive and / or negative selection based on cell markers. The cell markers of different T cell subsets are as described above. In a specific embodiment, a specific subset of T cells is separated by positive or negative selection techniques, such as cells that are positive or express high levels of one or more surface markers, for example CCR7, CD45RO, CD8, CD27, CD28, CD62L, CD127, CD4 and / or CD45RA T cells.

[0246] Anti-CD3 / anti-CD28 conjugated magnetic beads (e.g. M-450 CD3 / CD28 T cell expander) positively selects and expands CD3 + 、CD28 + T cells.

[0247] In certain embodiments, CD8 + or CD4 + Selection step for separation of CD4 +Helper T cells and CD8 + Cytotoxic T cells. This CD8 + and CD4 + The population can be further sorted into subpopulations by positive or negative selection for markers that are expressed or expressed at a relatively high level on one or more naive T cell, memory T cell and / or effector T cell subpopulations.

[0248] In some embodiments, central memory T (T CM In a specific embodiment, memory T cells are present in CD8 + PBMCs can be enriched or depleted for CD62L, CD8, and / or CD62L, such as by using anti-CD8 and anti-CD62L antibodies. + CD8 + part.

[0249] In some embodiments, the central memory T (T CM In some aspects, enrichment of T cells is based on positive or high surface expression of CCR7, CD45RO, CD27, CD62L, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, enriched T cells are isolated by depletion of cells expressing CD4, CD14, CD45RA and positive selection or enrichment of cells expressing CCR7, CD45RO, and / or CD62L. CM CD8 + In one aspect, the central memory T (T CM ) cells are enriched starting with a negative fraction of cells selected based on CD4 expression, which is then subjected to negative selection based on expression of CD14 and CD45RA and positive selection based on CD62L. Such selections are performed simultaneously in some aspects, and sequentially in either order in other aspects. In some aspects, the enrichment for CD8 + The same selection procedure based on CD4 expression of cell populations or subsets is also used to generate CD4 + Cell populations or subpopulations such that both positive and negative fractions from CD4-based separation are retained, optionally after one or more additional positive or negative selection steps.

[0250] In a specific example, a PBMC sample or other leukocyte sample is subjected to CD4 +The negative fraction is then subjected to negative selection based on the expression of CD14 and CD45RA or RORI, and positive selection is performed based on markers characteristic of central memory T cells such as CCR7, CD45RO and / or CD62L, wherein the positive and negative selections are performed in either order.

[0251] In certain embodiments, cell enrichment produces a large population of CD8+ FACS sorted cells.

[0252] The T cell population can be incubated in the culture initiation composition to expand the T cell population. The incubation can be carried out in a culture container, such as a bag, cell culture plate, flask, chamber, chromatographic column, cross-linked gel, cross-linked polymer, column, culture dish, hollow fiber, microtiter plate, silica-coated glass plate, tube, tube set, well, vial or other container for culturing or growing cells.

[0253] Culture conditions may include one or more of the following: specific culture media, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

[0254] In some aspects, incubation is performed according to techniques such as described in US 6,040,177, Klebanoff et al. (2012) J Immunother. 35(9):651-660, Terakura et al. (2012) Blood. 1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0255] Exemplary culture media for culturing T cells include (i) RPMI supplemented with nonessential amino acids, sodium pyruvate, and penicillin / streptomycin; (ii) RPMI with HEPES, 5-15% human serum, 1-3% L-glutamine, 0.5-1.5% penicillin / streptomycin, and 0.25×10 -4 -0.75x10 -4(iii) RPMI supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 10mM HEPES, 100U / ml penicillin and 100m / mL streptomycin; (iv) DMEM medium supplemented with 10% FBS, 2mM L-glutamine, 10mM HEPES, 100U / ml penicillin and 100m / mL streptomycin; and (v) X-Vivo 15 medium (Lonza, Walkersville, MD) supplemented with 5% human AB serum (Gemcell, West Sacramento, CA), 1% HEPES (Gibco, Grand Island, NY), 1% Pen-Strep (Gibco), 1% GlutaMax (Gibco) and 2% N-acetylcysteine ​​(Sigma-Aldrich, St. Louis, MO). T cell culture media can also be purchased from Hyclone (Logan, UT). Other T cell activation components that can be added to such culture media are described in more detail below.

[0256] In some embodiments, feeder cells such as non-dividing peripheral blood mononuclear cells (PBMC) are added to the culture starting composition (e.g., so that for each T lymphocyte in the starting population to be amplified, the resulting cell population contains at least 5, 10, 20 or 40 or more PBMC feeder cells); and incubating the culture (e.g., for a time sufficient to amplify the number of T cells). In some aspects, non-dividing feeder cells may include γ-irradiated PBMC feeder cells. In some embodiments, PBMCs are irradiated with gamma rays in the range of 3000 to 3600rads to prevent cell division. In some aspects, feeder cells are added to the culture medium before adding the T cell population.

[0257] Optionally, incubation can also include adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of 6000 to 10,000 rads. In some aspects, LCL feeder cells are provided in any suitable amount, such as a ratio of LCL feeder cells to naive T lymphocytes of at least 10:1.

[0258] In some embodiments, the stimulating conditions include a temperature suitable for the growth of human T lymphocytes, such as at least 25°C, at least 30°C, or 37°C.

[0259] The activation culture conditions of T cells include conditions under which the T cells of the starting composition are cultured to proliferate or expand.

[0260] (VI) Preparation and cryopreservation of cells. Cells genetically modified using a minimal manipulation manufacturing process can be directly administered to a subject after genetic modification. In a specific embodiment, the genetically modified cells can be formulated into a cell-based composition for administration to a subject. A cell-based composition refers to a cell prepared with a pharmaceutically acceptable carrier for administration to a subject.

[0261] Exemplary carriers and modes of cell administration are described in U.S. Patent Publication No. 2010 / 0183564, pages 14-15. Other drug carriers are described in Remington: The Science and Practice of Pharmacy, 21st edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005).

[0262] In certain embodiments, cells can be harvested from the culture medium, washed and concentrated into a carrier in a therapeutically effective amount. Exemplary carriers include saline, buffered saline, physiological saline, water, Hanks solution, Ringer solution, Nonnosol-R (Abbott Labs), Plasma-Lyte (Baxter Laboratories, Inc., Morton Grove, IL), glycerol, ethanol, and combinations thereof.

[0263] In a specific embodiment, the carrier can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In a specific embodiment, the carrier for infusion includes buffered saline with 5% HSA or dextrose. Other isotonic agents include polyols, including trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol or mannitol.

[0264] The carrier may include a buffer such as a citrate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a gluconate buffer, an oxalate buffer, a lactate buffer, an acetate buffer, a phosphate buffer, a histidine buffer, and / or a trimethylamine salt.

[0265] Stabilizers refer to a broad class of excipients whose functions can range from fillers to additives that help prevent cells from adhering to the walls of the container. Typical stabilizers can include polyols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclic alcohols such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as HSA, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran.

[0266] Where necessary or beneficial, the cell-based compositions may include a local anesthetic such as lidocaine to ease pain at the site of injection.

[0267] Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium chloride, hexamethylammonium chloride, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0268] For example, a therapeutically effective amount of cells in a cell-based composition may be greater than 10 2 cells, greater than 10 3 cells, greater than 10 4 cells, greater than 10 5 cells, greater than 10 6 cells, greater than 10 7 cells, greater than 10 8 cells, greater than 10 9 cells, greater than 10 10 cells or more than 10 11 If the patient is conditioned, a product equivalent to at least 2 million CD34+ cells / kg body weight is preferably infused. In unconditioned patients, at least 1 million CD34+ cells / kg body weight is acceptable.

[0269] In the cell-based compositions disclosed herein, the volume of cells is typically 1 liter or less, 500 mL or less, 250 mL or less, or 100 mL or less. Thus, the density of cells administered is typically greater than 104 cells / mL, 10 7 cells / mL or 10 8 cells / mL.

[0270] The cells or cell-based compositions disclosed herein can be prepared for administration by, for example, injection, infusion, perfusion or lavage. The cells or cell-based compositions can be further formulated for bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intracapsular and / or subcutaneous injection.

[0271] In a specific embodiment, the cell or cell-based composition is administered to a subject in need thereof as quickly as possible after the genetic modification and / or formulation administration is completed. In a specific embodiment, cryopreservation of cells may be necessary or beneficial. The terms "frozen / freezing" and "cryopreserved / cryopreserving" can be used interchangeably. Freezing includes freeze drying. In a specific embodiment, cryopreservation of fresh cells can reduce unwanted cell colonies. Therefore, a specific embodiment includes cryopreservation of biological samples before administering NP to the sample. In a specific embodiment, the biological sample is washed to remove platelets before cryopreservation.

[0272] As will be appreciated by those of ordinary skill in the art, freezing of cells can be destructive (see Mazur, P., 1977, Cryobiology 14: 251-272), but there are many methods available to prevent such damage. For example, damage can be avoided by (a) using cryoprotectants, (b) controlling the freezing rate, and / or (c) storing at sufficiently low temperatures to minimize degradation reactions. Exemplary cryoprotectants include dimethyl sulfoxide (DMSO) (Lovelock and Bishop, 1959, Nature 183:1394-1395; Ashwood-Smith, 1961, Nature 190:1204-1205), glycerol, polyvinyl pyrrolidine (Rinfret, 1960, Ann. NY Acad. Sci. 85:576), polyethylene glycol (Sloviter and Ravdin, 1962, Nature 196:548), albumin, dextran, sucrose, ethylene glycol, erythritol, D-ribitol, D-mannitol (Rowe et al., 1962, Fed. Proc. 21:157), D-sorbitol, i-inositol, D-lactose, choline chloride (Bender et al., 1960, J. Appl. Physiol. 15:520), amino acids (Phan The Tran and Bender, 1960, Exp. Cell Res. 20:651), methanol, acetamide, monoacetin (Lovelock, 1954, Biochem. J. 56:265) and inorganic salts (Phan The Tran and Bender, 1960, Proc. Soc. Exp. Biol. Med. 104:388; Phan The Tran and Bender, 1961, in Radiobiology, Proceedings of the Third Australian Conference on Radiobiology, llbery ed., Butterworth, London, p. 59). In certain embodiments, DMSO can be used. Adding plasma (e.g., to a concentration of 20-25%) can enhance the protective effect of DMSO. After adding DMSO, cells can be kept at 0°C until freezing, because 1% DMSO concentration can be toxic at temperatures above 4°C.

[0273] In cryopreservation of cells, a slowly controlled cooling rate may be critical, and different cryoprotectants (Rapatz et al., 1968, Cryobiology 5(1):18-25) and different cell types have different optimal cooling rates (see, e.g., Rowe and Rinfret, 1962, Blood 20:636; Rowe, 1966, Cryobiology 3(1):12-18; Lewis, et al., 1967, Transfusion 7(1):17-32; and Mazur, 1970, Science 168:939-949 for effects of cooling velocity on survival of stem cells and on their transplantation potential). The heat of fusion of water into ice should be minimal. The cooling process can be performed using, for example, a programmable freezing device or a methanol bath process. Programmable freezing equipment allows determination of the optimal cooling rate and facilitates standard, reproducible cooling.

[0274] In a specific embodiment, DMSO treated cells can be pre-cooled on ice and transferred to a tray containing chilled methanol, which is then placed in a mechanical freezer (e.g., Harris or Revco) at -80°C. Thermocouple measurements of the methanol bath and samples indicate that a cooling rate of 1°C / min to 3°C / min may be preferred. After at least two hours, the specimen can reach a temperature of -80°C and can be placed directly in liquid nitrogen (-196°C).

[0275] After complete freezing, the cells can be quickly transferred to long-term cryogenic storage containers. In certain embodiments, samples can be cryogenically stored in liquid nitrogen (-196°C) or vapor (-1°C). The availability of high-efficiency liquid nitrogen freezers facilitates this storage.

[0276] Additional considerations and procedures for manipulation, cryopreservation, and long-term storage of cells can be found in the following exemplary references: U.S. Pat. Nos. 4,199,022; 3,753,357; and 4,559,298; Gorin, 1986, Clinics In Haematology 15(1):19-48; Bone-Marrow Conservation, Culture and Transplantation, Proceedings of a Panel, Moscow, July 22-26, 1968, International Atomic Energy Agency, Vienna, pp. 107-186; Livesey and Linner, 1987, Nature 327:255; Linner et al., 1986, J. Histochem. Cytochem. 34(9):1123-1135; Simione, 1992, J. Parenter. Sci. Technol. 46(6):226-32).

[0277] After cryopreservation, the frozen cells can be thawed for use according to methods known to those of ordinary skill in the art. The frozen cells are preferably thawed quickly and frozen immediately after thawing. In a specific embodiment, the vial containing the frozen cells can be immersed in a warm water bath up to its neck; gentle rotation will ensure that the cell suspension is mixed as it thaws and increase heat transfer from the warm water to the internal ice. Once the ice is completely melted, the vial can be immediately placed on ice.

[0278] In certain embodiments, methods to prevent cells from clumping during thawing can be used. Exemplary methods include: adding DNase (Spitzer et al., 1980, Cancer 45:3075-3085), low molecular weight dextran and citrate, hydroxyethyl starch (Stiff et al., 1983, Cryobiology 20:17-24), etc. before and / or after freezing.

[0279] As will be appreciated by one of ordinary skill in the art, if a cryoprotectant is used that is toxic to humans, it should be removed prior to therapeutic use. DMSO is not severely toxic.

[0280] (VII) Nanoparticle Formulations. The NPs disclosed herein can also be formulated for direct administration to a subject. Figure 4As shown, the size of AuNPs can be selected to affect biodistribution in the human body.NPs suitable for the present disclosure can be of any shape and can range in size from 5nm-1000nm, such as 5nm-10nm, 5-50nm, 5nm-75nm, 5nm-40nm, 10nm-30 or 20nm-30nm. The size of the NPs may also be in the following ranges: 10nm-15nm, 15nm-20nm, 20nm-25nm, 25nm-30nm, 30nm-35nm, 35nm-40nm, 40nm-45nm, or 45nm-50nm, 50nm-55nm, 55nm-60nm, 60nm-65nm, 65nm-70nm, 70nm-75nm, 75nm-80nm, 80nm-85nm, 85nm-90nm, 90nm-95nm, 95nm-100nm, 100nm-105nm, 105nm-106nm. In some embodiments, NPs with a size of 550 nm or more are excluded. This is because particles with a size of >600 nm or aggregated particles are not suitable for cellular uptake.

[0281] A therapeutically effective amount of NPs in the composition may include at least 0.1% w / v or w / w particles; at least 1% w / v or w / w particles; at least 10% w / v or w / w particles; at least 20% w / v or w / w particles; at least 30% w / v or w / w particles; at least 40% w / v or w / w particles; at least 50% w / v or w / w particles; at least 60% w / v or w / w particles; at least 70% w / v or w / w particles; at least 80% w / v or w / w particles; at least 90% w / v or w / w particles; at least 95% w / v or w / w particles; or at least 99% w / v or w / w particles.

[0282] (VIII) Kits. The present disclosure also provides kits comprising any one or more of the elements disclosed herein. In specific embodiments, the kit may comprise NPs as described herein, comprising guide RNAs and nucleases capable of cleaving target sequences. The kit may additionally comprise one or more HDTs, targeting ligands and / or polymers (e.g., PEG, PEI). The elements may be provided alone or in combination, and may be provided in any suitable container, such as a vial, bottle, bag, or tube. In some embodiments, the kit comprises instructions in one or more languages.

[0283] In a specific embodiment, the kit comprises one or more reagents used in the method of obtaining one or more of the elements described herein. The reagent can be provided in any suitable container. For example, the kit can provide one or more reaction or storage buffers. The reagent can be provided in a form that can be used for a specific assay or in a form that requires the addition of one or more other components before use (for example, in a concentrate or lyophilized form). The buffer can be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of 7 to 10. In some embodiments, the kit comprises a guide RNA (e.g., cRNA), a nuclease (e.g., Cpf1), an Au core and / or a homologous recombination template polynucleotide.

[0284] The kit may also include one or more components to collect, process, modify and / or prepare cells for administration. The kit may provide components for in vitro cell manufacturing with reduced or minimal manipulation. It may also include a manufactured article and / or instructions for clinical staff.

[0285] (IX) Exemplary Methods of Use. As noted, the selected cell type can be obtained from a subject. In certain embodiments, the cells are reintroduced in a therapeutically effective amount into the same subject from which the original sample was derived. In certain embodiments, the cells are administered in a therapeutically effective amount to a different subject.

[0286] The compositions and formulations disclosed herein can be used to treat subjects (humans, veterinary animals (dogs, cats, reptiles, birds, etc.), livestock (horses, cows, goats, pigs, chickens, etc.), and research animals (monkeys, rats, mice, fish, etc.). In specific embodiments, the subject is a human patient.

[0287] Examples of diseases that can be treated using the NP compositions or cell preparations described herein that are made with reduced or minimal manipulation include monogenic blood disorders, hemophilia, Graves' disease, rheumatoid arthritis, pernicious anemia, multiple sclerosis (MS), inflammatory bowel disease, systemic lupus erythematosus (SLE), Wiskott-Aldrich syndrome (WAS), chronic granulomatous disease (CGD), Battens disease, adrenoleukodystrophy (ALD) or metachromatic leukodystrophy (MLD), muscular dystrophy, alveolar proteinosis (PAP), pyruvate kinase deficiency, Shwachmann-Diamond-Blackfan anemia, dyskeratosis congenita, cystic fibrosis, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myeloid metaplasia of unknown cause, megakaryocytosis / congenital thrombocytopenia, ataxia-telangiectasia, thalassemia major, CLL, chronic myeloid leukemia (CML), chronic myelomonocytic leukemia, common variable immunodeficiency (CVID), complement disorders, congenital (X-linked) agammaglobulinemia, familial erythrophagocytic lymphohistiocytosis, Hodgkin lymphoma, Hurler syndrome, hyper-IgM, IgG subclass deficiency, juvenile myelomonocytic leukemia, mucopolysaccharidosis, multiple myeloma, myelodysplasia, non-Hodgkin lymphoma, paroxysmal nocturnal hemoglobinuria (PNH), primary immunodeficiency with antibody deficiency, pure red cell aplasia, refractory anemia, selective IgA deficiency, severe aplastic anemia, SCD and / or specific antibody deficiency.

[0288] (X) Exemplary manufacturing embodiments and comparisons.

[0289]

[0290] Comparison of exemplary fabrication schemes.

[0291]

[0292]

[0293]

[0294] (XI) Assays to evaluate nanoparticle performance. Assays known in the art can be used to evaluate the effectiveness of the NPs described herein, including: the effectiveness of NP uptake by a cell population, the effect of NP uptake on cell viability, and any residual presence of NPs in a minimally manipulated blood cell product (including a cell population genetically modified using the NPs described herein). As described above, the presence, level, or rate of gene editing of a selected cell population can also be determined. Assays can also be used to determine whether to select a therapeutic formulation containing the NPs described herein and / or whether to select a minimally manipulated blood cell product containing a cell population genetically modified using the NPs described herein for further development.

[0295] The uptake of NP by cell populations can be evaluated by various methods known in the art, including confocal microscopy, fluorescence activated cell sorting (FACS) and inductively coupled plasma (ICP) technology, including: ICP-mass spectrometry (ICP-MS), ICP-atomic emission spectroscopy (ICP-AES) and ICP-optical emission spectroscopy (ICP-OES). In a specific embodiment, crRNA and / or donor templates can be labeled with dyes and evaluated for cell uptake using confocal microscopy. In a specific embodiment, FACS using fluorescently labeled antibodies that recognize cell surface markers can be used in conjunction with confocal microscopy to test whether the target cell population has been targeted by labeled NPs. In a specific embodiment, labeled antibodies that recognize cell surface markers are on small magnetized particles, and immunomagnetic bead-based sorting can be performed to determine that the cell population has been targeted by labeled NPs. In a specific embodiment, ICP technology allows qualitative and quantitative trace element detection. A specific embodiment of ICP uses plasma to atomize or excite samples for detection. In a specific embodiment, ICP can be generated by directing the energy of a radio frequency generator into a suitable gas such as ICP argon, helium or nitrogen. In certain embodiments, ICP-MS can be used to detect any residual NPs in a minimally manipulated blood cell product comprising a population of cells genetically modified using the NPs described herein.

[0296] In specific embodiments, 50% to 100%, 50% to 90% or 50% to 80% of the target cells take up the NPs described herein. In specific embodiments, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the target cells take up the NPs described herein. In specific embodiments, the target cells are cells targeted for genetic modification by the NPs described herein. In specific embodiments, the target cells are cells targeted by the NPs through targeting ligands on the NPs that bind to cell surface markers on the cells. In specific embodiments, non-target cells are cells that are not targeted for genetic modification by the NPs described herein. In specific embodiments, non-target cells are cells that are not targeted by the NPs described herein because they do not express cell surface markers recognized by the targeting ligands on the NPs.

[0297] Cell viability after treatment with Au / CRISPR NP can be analyzed at different time points using trypan blue, a dye that specifically marks dead cells and can therefore be used to distinguish live cells from dead cells. Trypan blue is available from commercial distributors such as Invitrogen (Carlsbad, CA). Cell counting can be performed using a cell counter such as the Countess II FL automatic cell counter from ThermoFisherScientific (Waltham, MA). The cell viability percentage of each sample can be recorded and reported as mean ± SD.

[0298] Fluorescence-based assays such as those from Invitrogen (Carlsbad, CA) can also be used. Assay kit to analyze cell viability, in In the assay, two compounds can distinguish between live and dead cells. First, dyes that do not permeate cells (e.g., ethidium homodimer-1) only bind to the surface of live cells and produce very weak fluorescence, while dyes can penetrate the cell membrane of dead cells and bind to internal molecules, producing very bright fluorescence. Second, non-fluorescent cell permeable dyes (e.g., calcein AM) can be converted into a strongly fluorescent form (e.g., calcein) by esterase activity in live cells. The labeled cells can be imaged under a fluorescence microscope using appropriate excitation and emission values. Live and dead cells can be counted and imaged using appropriate software.

[0299] In specific embodiments, 70% to 100%, 70% to 90%, or 70% to 80% of the target cells are alive after treatment with a therapeutic formulation comprising the NPs described herein. In specific embodiments, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the target cells are alive after treatment with a therapeutic formulation comprising the NPs described herein.

[0300] In a specific embodiment, the fitness of HSC / HSPC treated with NPs described herein can be assessed by colony forming cell (CFC) assay (also referred to as methylcellulose assay). In the CFC assay, the ability of HSC / HSPC to proliferate and differentiate into colonies in a semisolid culture medium in response to cytokine stimulation can be assessed. Cells can be plated in methylcellulose containing recombinant human growth factor and incubated for a specified period of time. The resulting colonies can be counted and the morphology can be scored on a stereomicroscope to determine the number of colony-forming cells per plated cell number (e.g., 100,000 plated cells).

[0301] In certain embodiments, the fitness of HSC / HSPC treated with NPs described herein can be assessed by in vivo studies using sublethally irradiated immunodeficient (NOD / SCIDγ- / -; NSG) mice. These studies can assess the fitness of HSC / HSPC by the ability of the cells to reconstitute the myelosuppressive host. In certain embodiments, a specified number of cells can be infused into NSG mice, and the mice can be followed for several weeks to assess the engraftment of HSC / HSPC.

[0302] Implantation of HSC / HSPC and / or other cell colonies can be assessed by collecting biological samples (e.g., blood, bone marrow, spleen) from mice and performing FACS using fluorescent marker antibodies in conjunction with cell surface markers. In specific embodiments, FACS can detect the level of cells (HSC / HSPC) expressing CD45, cells (B cells) expressing CD20, cells (monocytes) expressing CD14, cells (T cells) expressing CD3, cells (T cells) expressing CD4, and cells (T cells) expressing CD8. In specific embodiments, sorting based on immunomagnetic beads can be used that includes small magnetized particles containing antibodies in conjunction with cell surface markers.

[0303] In a specific embodiment, the therapeutic formulations comprising the NPs described herein can be subjected to release testing to determine the suitability of the therapeutic formulation for in vivo re-infusion testing. In a specific embodiment, the release testing includes Gram staining, 3-day sterility, 14-day sterility, mycoplasma, endotoxin, and cell viability by trypan blue. In a specific embodiment, if the release test produces: negative results for Gram staining, 3-day sterility, 14-day sterility, and mycoplasma; ≤0.5EU / mL endotoxin; and ≥70% viability by trypan blue, the therapeutic formulation can be advanced for further development.

[0304] In certain embodiments, the performance of minimally manipulated blood cell products comprising a cell population genetically modified using the NPs described herein can be evaluated in vivo using NSG mice.In certain embodiments, engraftment of HSC / HSPC and / or other cell populations can be evaluated as described above.

[0305] According to the scheme described in Burkholder et al. Health Evaluation of Experimental Laboratory Mice. Current Protocols in Mouse Biology, 2012; 2: 145-165, mice infused with minimally manipulated blood cell products containing a cell population genetically modified using NPs described herein can be visually monitored for any effects on health (e.g., grooming, weight, activity level). In a specific embodiment, the presence of NPs in the infused blood cell products can be assessed by ICP-MS. In a specific embodiment, the presence of NPs in the urine and feces of mice can be assessed by ICP-MS at a given time (e.g., 72 hours) after infusion to determine whether all NPs have been cleared (mass balance). In a specific embodiment, the minimum threshold in urine / feces within 72 hours is 0, and the maximum threshold cannot exceed the total mass of the injection. If bioaccumulation is indicated, micro-computed tomography (CT) imaging of live mice can be performed to assess the location of accumulation. In a specific embodiment, ICP-MS and / or autopsy can also be performed to determine the location of bioaccumulation. In a specific embodiment, micro-CT, autopsy and / or trace element analysis (e.g., ICP-MS) can be combined with histopathology to assess the potential toxicity of NP in infused mice. In a specific embodiment, organ toxicity in infused mice is compared with untreated controls from all donors. In a specific embodiment, for histopathology, the minimum threshold is non-toxic, and the maximum threshold is graded using the adverse event criteria published for each target organ.

[0306] (XII) Exemplary embodiments.

[0307] 1. A method for genetically modifying a selected cell population in a biological sample that has undergone reduced or minimal manipulation, comprising adding a nanoparticle (NP) disclosed herein to the biological sample.

[0308] 2. The method of embodiment 1, wherein the NP is a gold NP (AuNP).

[0309] 3. A method as described in embodiment 1 or 2, wherein the NP comprises a guide RNA (gRNA), wherein one end of the gRNA is conjugated to a linker and the other end of the gRNA is conjugated to a nuclease, and wherein the linker allows the gRNA to be covalently attached to the surface of the NP.

[0310] 4. A method as described in embodiment 3, wherein the gRNA comprises a clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA (crRNA).

[0311] 5. A method as described in embodiment 4, wherein the 3' end of the crRNA is conjugated to the linker.

[0312] 6. A method as described in embodiment 4, wherein the 5' end of the crRNA is conjugated to the linker.

[0313] 7. A method as described in embodiment 4 or 5, wherein the 5' end of the crRNA is conjugated to the nuclease.

[0314] 8. A method as described in embodiment 4 or 6, wherein the 3' end of the crRNA is conjugated to the nuclease.

[0315] 9. The method of any one of embodiments 3-8, wherein the linker comprises a spacer having a thiol modification.

[0316] 10. The method of embodiment 9, wherein the spacer is an oligoethylene glycol spacer.

[0317] 11. The method of embodiment 10, wherein the oligoethylene glycol spacer is a 10-26 atom oligoethylene glycol spacer.

[0318] 12. The method of embodiment 10 or 11, wherein the oligoethylene glycol spacer is an 18-atom oligoethylene glycol spacer.

[0319] 13. A method as described in any one of embodiments 3-12, wherein the crRNA comprises a sequence as shown in SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:13; SEQ ID NO:14; or SEQ ID NO:225-264.

[0320] 14. The method of any one of embodiments 3-13, wherein the NP further comprises a donor template that is farther away from the surface of the NP than the gRNA and the nuclease.

[0321] 15. The method of embodiment 14, wherein the donor template comprises a therapeutic gene.

[0322] 16. A method as described in embodiment 15, wherein the therapeutic gene comprises or encodes skeletal protein 4.1, glycophorin, p55, Duffy allele, globin family gene; WAS; phox; dystrophin; pyruvate kinase; CLN3; ABCD1; arylsulfatase A; SFTPB; SFTPC; NLX2.1; ABCA3; GATA1; ribosomal protein gene; TERT; TERC; DKC1; TINF2; CFTR; LRRK2; PARK2; PARK7; PINK1; SNCA; PSEN1; PSEN2; APP; SOD1; TDP43; FUS; ubiquitin 2; C9ORF72, α2β1; αvβ3; αvβ5; αvβ63; BOB / GPR15; Bonzo / STRL-33 / TYMSTR; CCR2; CCR3; CCR5; 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, ApoAIV, ApoE, ATM, BAI-1, BDNF, B eta*(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, e rb, ERBA, ERBB, ETS1, ETS2, ETV6, Fab, FancA, FancB, FancC, FancDI, FancD2, FancE, FancF, FancG, Fancl, FancJ, FancL, FancM, FancN, FancO, FancP, F ancQ, FancR, FancS, FancT, FancU, FancV, and FancW, FCC, FGF, FGR, FHIT, fms, FOX, FUS1, 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 alpha, interferon beta, interferon gamma, 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, 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. ,

[0323] 17. The method of any one of embodiments 14-16, wherein the donor template comprises a homology directed repair template (HDT) comprising a sequence having homology to the genomic sequence undergoing modification.

[0324] 18. The method of embodiment 18, wherein the HDT comprises a sequence as shown in SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:8; SEQ ID NO:15; SEQ ID NOs:33-41 or SEQ ID NOs:44-52.

[0325] 19. The method of any one of embodiments 14-18, wherein the donor template comprises single-stranded DNA (ssDNA).

[0326] 20. A method as described in any of embodiments 1-19, wherein the NP is an AuNP associated with at least three layers, wherein the first layer comprises single-stranded DNA (ssDNA), the second layer comprises clustered regularly interspaced short palindromic repeats (CRISPR) guide RNA (crRNA), and the third layer comprises a nuclease, and wherein the first layer is the layer closest to the surface of the AuNP core, the second layer is the layer second closest to the surface of the AuNP core, and the third layer is the layer third closest to the surface of the AuNP core.

[0327] 21. The method of embodiment 20, wherein the first layer further comprises polyethylene glycol (PEG).

[0328] 22. The method of any one of embodiments 1-21, wherein the added amount is 1, 2, 3, 4, 5, 8, 10, 12, 15, or 20 μg NP per milliliter (mL) of biological sample.

[0329] 23. The method of any one of embodiments 1-22, wherein the biological sample and the added NPs are incubated for 1-48 hours.

[0330] 24. The method of any one of embodiments 1-22, wherein the biological sample and the added NPs are incubated until testing confirms uptake of the NPs into cells.

[0331] 25. The method of embodiment 24, wherein the testing comprises confocal microscopy imaging or inductively coupled plasma (ICP) technology.

[0332] 26. A method as described in embodiment 24 or 25, wherein the testing comprises ICP-mass spectrometry (ICP-MS), ICP-atomic emission spectrometry (ICP-AES) or ICP-optical emission spectrometry (ICP-OES).

[0333] 27. The method of any one of embodiments 1-26, wherein the NPs are associated with a positively charged polymer (e.g., polyethyleneimine (PEI)) coating.

[0334] 28. A method as described in embodiment 27, wherein the positively charged polymer coating produces the surface of the NP, wherein the surface optionally comprises a donor template.

[0335] 29. The method of any one of embodiments 1-28, wherein the NP comprises a targeting ligand.

[0336] 30. The method of embodiment 29, wherein the targeting ligand comprises an antibody or antigen-binding fragment thereof, an aptamer, a protein and / or a binding domain.

[0337] 31. The method of embodiment 29 or 30, wherein the targeting ligand extends beyond the surface of the NP.

[0338] 32. A method as described in any one of embodiments 29-31, wherein the targeting ligand is a binding molecule that binds to CD3, CD4, CD34, CD46, CD90, CD133, CD164, luteinizing hormone releasing hormone (LHRH) receptor or aryl hydrocarbon receptor (AHR) (e.g., antibody clone: ​​581; antibody clone: ​​561; antibody clone: ​​REA1164; 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).

[0339] 33. The method of any one of embodiments 29-32, wherein the targeting ligand is an anti-human CD3 antibody or an antigen-binding fragment thereof, an anti-human CD4 antibody or an antigen-binding fragment thereof, an anti-human CD34 antibody or an antigen-binding fragment thereof, an anti-human CD46 antibody or an antigen-binding fragment thereof, an anti-human CD90 antibody or an antigen-binding fragment thereof, an anti-human CD133 antibody or an antigen-binding fragment thereof, an anti-human CD164 antibody or an antigen-binding fragment thereof, an anti-human CD133 aptamer, human luteinizing hormone, human chorionic gonadotropin, degaerolide acetate, or StemRegenin 1.

[0340] 34. The method of any one of embodiments 29-33, wherein the nuclease and targeting ligand are linked.

[0341] 35. A method as described in embodiment 34, wherein the nuclease and the targeting ligand are connected by an amino acid linker (e.g., a direct amino acid linker, a flexible amino acid linker, or a tag-based amino acid linker (e.g., a Myc tag or a Strep tag)).

[0342] 36. A method as described in embodiment 34 or 35, wherein the nuclease and targeting ligand are linked by polyethylene glycol.

[0343] 37. The method of any one of embodiments 34-36, wherein the nuclease and the targeting ligand are linked via an amine-thiol crosslinker.

[0344] 38. The method of any one of embodiments 3-37, wherein the nuclease is selected from Cpf1, Cas9, or Mega-TAL.

[0345] 39. The method of any one of embodiments 3-38, wherein the nuclease is Cpf1.

[0346] 40. The method of any one of embodiments 34-39, wherein the targeting ligand attached to the nuclease is further away from the surface of the NP than the ssDNA associated with the NP.

[0347] 41. The method of any one of embodiments 1-40, wherein the NP is associated with a crRNA targeting a site described herein.

[0348] 42. The method of any one of embodiments 1-41, wherein the method targets a genomic site comprising a sequence selected from a sequence comprising SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NOs: 20-32; SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NOs: 84-97; or SEQ ID NOs: 214-224.

[0349] 43. The method of any one of embodiments 1-42, wherein the method comprises targeting a genomic site for genetic modification with a sequence selected from SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 13; SEQ ID NO: 14; or SEQ ID NOs: 225-264.

[0350] 44. A method as described in any of embodiments 1-43, wherein the selected cell population includes blood cells selected from the following: hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), hematopoietic stem and progenitor cells (HSPC), T cells, natural killer (NK) cells, B cells, macrophages, monocytes, mesenchymal stem cells (MSC), white blood cells (WBC), mononuclear cells (MNC), endothelial cells (EC), stromal cells and / or bone marrow fibroblasts.

[0351] 45. The method of embodiment 44, wherein the blood cells include CD34 + CD45RA - CD90 + HSC.

[0352] 46. ​​The method of embodiment 44 or 45, wherein the blood cells include CD34 + / CD133 + HSC.

[0353] 47. The method of any one of embodiments 44-46, wherein the blood cells include LH + HSC.

[0354] 48. The method of any one of embodiments 44-47, wherein the blood cells include CD34 + CD90 + HSPC.

[0355] 49. The method of any one of embodiments 44-48, wherein the blood cells include CD34 + CD90 + CD133 + HSPC.

[0356] 50. The method of any one of embodiments 44-49, wherein the blood cells include AHR + HSPC.

[0357] 51. The method of any one of embodiments 44-50, wherein the blood cells include CD3 + T cells.

[0358] 52. The method of any one of embodiments 44-51, wherein the blood cells include CD4 + T cells.

[0359] 53. The method of any one of embodiments 44-52, wherein the blood cells are human blood cells.

[0360] 54. The method of any one of embodiments 1-53, wherein the biological sample comprises peripheral blood and / or bone marrow.

[0361] 55. The method of any one of embodiments 1-54, wherein the biological sample comprises granulocyte colony stimulating factor (GCSF) mobilized peripheral blood and / or plerixafor mobilized peripheral blood.

[0362] 56. The method of any one of embodiments 1-55, wherein the method produces an average overall gene editing rate of 5% to 50%.

[0363] 57. The method of any one of embodiments 1-56, wherein the method produces greater than 60% cell viability in a selected cell population.

[0364] 58. A cell modified according to the method of any one of embodiments 1-57.

[0365] 59. The cell of embodiment 58, wherein the cell has not undergone electroporation.

[0366] 60. The cell of embodiment 58 or 59, wherein the cell has not been exposed to a viral vector.

[0367] 61. The cell of any one of embodiments 58-60, wherein the cell has not been exposed to a viral vector encoding a donor template or HDT.

[0368] 62. The cell of any one of embodiments 58-61, wherein the cell has not undergone a cell separation process intended to isolate the cell from a biological sample.

[0369] 63. The cell of any one of embodiments 58-62, wherein the cell has not undergone a magnetic cell separation process.

[0370] 64. A therapeutic preparation comprising the cells of any one of embodiments 58-63.

[0371] 65. A method of providing a therapeutic nucleic acid sequence to a subject in need thereof, comprising administering to the subject a cell as described in any one of embodiments 58-63 or a therapeutic formulation as described in embodiment 64, thereby providing the subject with a therapeutic nucleic acid sequence

[0372] 66. A nanoparticle (NP) comprising:

[0373] The core diameter is less than 30nm;

[0374] 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 having a chemical modification and the 5' end is conjugated to the nuclease, and wherein the chemical modification is covalently attached to a surface of the core;

[0375] 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 core; and

[0376] A donor template (eg, optionally including a homology directed repair template (HDT)) is provided on the surface of the positively charged polymer coating.

[0377] 67. The NP of embodiment 66, wherein the core comprises gold (Au).

[0378] 68. The NP of embodiment 66 or 67, wherein the weight / weight (w / w) ratio of core to nuclease is 0.6.

[0379] 69. The NP of any one of embodiments 66-68, wherein the w / w ratio of the core to HDT is 1.0.

[0380] 70. The NP of any one of embodiments 66-69, wherein the NP has a diameter of less than 70 nm.

[0381] 71. The NP of any one of embodiments 66-70, wherein the NP has a polydispersity index (PDI) less than 0.2.

[0382] 72. The NP of any one of embodiments 66-71, wherein the gRNA comprises a clustered regularly interspaced short palindromic repeats (CRISPR) crRNA.

[0383] 73. The NP of embodiment 72, wherein the crRNA comprises a sequence as shown in SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO: 13; SEQ ID NO: 14; or SEQ ID NO: 225-264.

[0384] 74. The NP of any one of embodiments 66-73, wherein the nuclease comprises Cpf1 or Cas9.

[0385] 75. The NP of any one of embodiments 66-74, wherein the positively charged polymer coating comprises polyethyleneimine (PEI), polyamidoamine (PAMAM); polylysine (PLL), polyarginine; cellulose, dextran, spermine, spermidine, or poly(vinylbenzyltrialkylammonium).

[0386] 76. The NP of any one of embodiments 66-75, wherein the positively charged polymer has a molecular weight of 1500-2500 Daltons.

[0387] 77. The NP of any one of embodiments 66-76, wherein the positively charged polymer has a molecular weight of 2000 Daltons.

[0388] 78. The NP of any one of embodiments 66-77, wherein the chemical modification comprises a free thiol, amine, or carboxylate functional group.

[0389] 79. The NP of any one of embodiments 66-78, wherein the spacer comprises an oligoethylene glycol spacer.

[0390] 80. The NP of embodiment 79, wherein the oligoethylene glycol spacer comprises an 18-atom oligoethylene glycol spacer.

[0391] 81. The NP of any one of embodiments 66-80, wherein the HDT comprises a sequence having homology to a genomic sequence undergoing modification.

[0392] 82. The NP of embodiment 81, wherein the HDT comprises a sequence as shown in SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 8; SEQ ID NO: 15; SEQ ID NOs: 33-41; or SEQ ID NOs: 44-52.

[0393] 83. The NP of any one of embodiments 66-82, wherein the HDT comprises single-stranded DNA (ssDNA).

[0394] 84. The NP of any one of embodiments 66-83, wherein the donor template comprises a therapeutic gene.

[0395] 85. The NP of embodiment 84, wherein the therapeutic gene encodes skeletal protein 4.1, glycophorin, p55, Duffy allele, globin family gene; WAS; phox; dystrophin; pyruvate kinase; CLN3; ABCD1; arylsulfatase A; SFTPB; SFTPC; NLX2.1; ABCA3; GATA1; ribosomal protein gene; TERT; TERC; DKC1; TINF2; CFTR; LRRK2; PARK2; PARK7; PINK1; SNCA; PSEN1; PSEN2; APP; SOD1; TDP43; FUS; ubiquitin 2; C9ORF72, α2β1; αvβ3 ;αvβ5;αvβ63;BOB / GPR15;Bonzo / STRL-33 / TYMSTR;CCR2;CCR3;CCR5;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, 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, ERB A. ERBB, ETS1, ETS2, ETV6, Fab, FancA, FancB, FancC, FancDI, FancD2, FancE, FancF, FancG, Fancl, FancJ, FancL, FancM, FancN, FancO, FancP, FancQ, F ancR, FancS, FancT, FancU, FancV, and FancW, FCC, FGF, FGR, FHIT, fms, FOX, FUS1, 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 alpha, interferon beta, interferon gamma, 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, 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. ,

[0396] 86. The NP of any one of embodiments 66-85, wherein the NP further comprises a targeting ligand attached to the nuclease.

[0397] 87. The NP of embodiment 86, wherein the targeting ligand comprises a binding molecule that binds to CD3, CD4, CD34, CD46, CD90, CD133, CD164, luteinizing hormone-releasing hormone (LHRH) receptor, or aryl hydrocarbon receptor (AHR).

[0398] 88. The NP of embodiment 86 or 87, wherein the targeting ligand comprises an anti-human CD3 antibody or an antigen-binding fragment thereof, an anti-human CD4 antibody or an antigen-binding fragment thereof, an anti-human CD34 antibody or an antigen-binding fragment thereof, an anti-human CD46 antibody or an antigen-binding fragment thereof, an anti-human CD90 antibody or an antigen-binding fragment thereof, an anti-human CD133 antibody or an antigen-binding fragment thereof, an anti-human CD164 antibody or an antigen-binding fragment thereof, an anti-human CD133 aptamer, human luteinizing hormone, human chorionic gonadotropin, degaerolide acetate, or StemRegenin 1.

[0399] 89. The NP of embodiments 86-88, wherein the targeting ligand comprises antibody clone: ​​581; antibody clone: ​​561; antibody clone: ​​REA1164; 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.

[0400] 90. The NP of any one of embodiments 86-89, wherein the nuclease and targeting ligand are linked.

[0401] 91. The NP of embodiment 90, wherein the nuclease and the targeting ligand are connected via an amino acid linker (e.g., a direct amino acid linker, a flexible amino acid linker, or a tag-based amino acid linker).

[0402] 92. The NP of any one of embodiments 86-91, wherein the nuclease and the targeting ligand are linked via polyethylene glycol (PEG).

[0403] 93. The NP of any one of embodiments 86-92, wherein the nuclease and targeting ligand are linked via an amine-thiol crosslinker.

[0404] 94. A composition comprising the NPs of claims 66-93 and a biological sample.

[0405] 95. The composition of embodiment 94, wherein the biological sample comprises a selected cell population.

[0406] 96. A composition as described in embodiment 95, wherein the selected cell population includes blood cells selected from the following: hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), hematopoietic stem and progenitor cells (HSPC), T cells, natural killer (NK) cells, B cells, macrophages, monocytes, mesenchymal stem cells (MSC), white blood cells (WBC), mononuclear cells (MNC), endothelial cells (EC), stromal cells and / or bone marrow fibroblasts.

[0407] 97. The composition of embodiment 95, wherein the blood cells include CD34 + CD45RA - CD90 + HSC;CD34 + / CD133 +HSC; LH + HSC;CD34 + CD90 + HSPC;CD34 + CD90 + CD133 + HSPC; and / or AHR + HSPC.

[0408] 98. The composition of embodiment 95, wherein the blood cells include CD3 + T cells and / or CD4 + T cells.

[0409] 99. The composition of any one of embodiments 94-98, wherein the biological sample comprises peripheral blood, bone marrow, granulocyte colony stimulating factor (GCSF) mobilized peripheral blood, and / or plerixafor mobilized peripheral blood.

[0410] 100. The composition of any one of embodiments 94-99, wherein the amount of NPs in the biological sample is 1, 2, 3, 4, 5, 8, 10, 12, 15, or 20 μg NPs per milliliter (mL) of biological sample.

[0411] 101. A kit comprising one or more components as described in any one of the preceding embodiments.

[0412] (XIII) Experimental Examples. Example 1. Synthesis of gold nanoparticle cores. Gold nanoparticles (AuNPs) in the 15 nm size range were synthesized using a slightly modified Turkevich method. Turkevich et al., (1951). Discussions of the Faraday Society 11(0):55-75. A 0.25 mM chloroauric acid solution was brought to boiling point and reduced by adding a 3.33% sodium citrate solution and vigorously stirred for 10 minutes under a reflux system. The synthesized NPs were washed three times and redispersed in high purity water.

[0413] Cpf1 and Cas9 guide RNA structures. A single Cpf1 guide RNA was ordered from a commercial source (Integrated DNA Technologies; IDT) with two custom modifications at the 3' end. The first modification included an 18-atom oligoethylene glycol (OEG) spacer (iSp18), and the second modification included a thiol modification. The OEG spacer (e.g., polyethylene glycol (PEG) or hexaethylene glycol (HEG), etc.) was added at a ratio of 1 per oligonucleotide and was used to prevent electrostatic repulsion between the oligonucleotides. Although an 18-atom spacer was used, other lengths are also suitable. The thiol modification was also added at a ratio of 1 per oligonucleotide and was used as the basis for covalent interactions that bind the oligonucleotide to the AuNP surface.

[0414] 5'- / AltR1 / rUrA rArUrU rUrCrU rArCrU rCrUrU rGrUrA rGrAr U rCrArCrCrCrG rArUrC rCrArC rUrGrG rGrGrA rGrCrA / iSp18 / / 3ThioMC3-D / -3'(SEQ ID NO:5)

[0415] For cas9, a two-part guide system comprising tracrRNA and crRNA was used. The crRNA for Cas9 was ordered from IDT with the same 18-spacer-thiol modification as above, but at the 5' end.

[0416] 5'- / 5ThioMC6-D / / iSp18 / rCrA rCrCrC rGrArU rCrCrA rCrUrG rGrGrG rArGrCrGrUrU rUrUrA rGrArG rCrUrA rUrGrC rU / AltR2 / -3'(SEQ ID NO:6)

[0417] The accompanying tracrRNA is unmodified. In these sequences, the "r" stands for RNA, and a spacer is provided to facilitate reading.

[0418] Prepare Au / CRISPR NPs. Use crRNA modified with 18 spacers-thiol. Add AuNPs at a concentration of 10 μg / mL to the crRNA solution at an AuNP / crRNA w / w ratio of 0.5. Then, add citrate buffer at a pH of 3 at a concentration of 10 mM and mix for 5 minutes. The prepared AuNP / crRNA nanoconjugates are centrifuged and redispersed in PBS. Then, Cpf1 nuclease is added at an AuNP / Cpf1 w / w ratio of 0.6. Add 2000 MW of polyethyleneimine (PEI) at a concentration of 0.005% and mix thoroughly. In the final step, add ssDNA template at an AuNP / ssDNA w / w ratio of 1.

[0419] Example 2. Targeted homology-directed repair in hematopoietic stem and progenitor cells using highly efficient gene-editing nanoparticles. Abstract. Ex vivo CRISPR gene editing in hematopoietic stem and progenitor cells corrects genetic diseases, protects against infectious diseases, and provides new treatments for cancer. Although current approaches for gene editing using homologous recombination, electroporation followed by non-integrating viral transduction have resulted in high levels of gene editing at certain genetic loci, this complex manipulation results in cytotoxicity and compromised fitness of transplanted blood cells. Here, highly efficient gene-editing NPs were developed using colloidal AuNPs. To ensure that all required machinery is delivered upon uptake of a single NP, a payload design was developed that enables passive entry into cells without the need for electroporation or viruses. This small, highly monodisperse NP avoids lysosomal entrapment and successfully localizes to the nucleus in primary human hematopoietic stem and progenitor cells without observable toxicity. NP-mediated gene editing is efficient and maintained at multiple loci of therapeutic interest with different gene-editing nucleases. NP-treated primary cells have better engraftment kinetics in humanized mice relative to untreated cells, with no observable differences in in vivo differentiation. This is the first demonstration of efficient passive delivery of an entire gene editing payload into primary human blood stem and progenitor cells.

[0420] Introduction. Retroviral-mediated gene correction in hematopoietic stem and progenitor cells (HSPCs) has demonstrated therapeutic results for a variety of genetic, infectious, and malignant conditions (Hacein-Bey-Abina et al., N Engl J Med, 371(15):1407-1417 (2014); Cicalese et al., Blood, 128(1):45-54 (2016); Sessa et al., Lancet, 388(10043):476-487 (2016); Hacein-Bey et al., JAMA, 313(15):1550-1563 (2015); and Dunbar et al., Science, 359(6372) (2018)), and the use of genetically modified autologous or "self" HSPCs eliminates the risk of graft-host immune response and the need for immunosuppressive drugs required for allogeneic hematopoietic stem cell transplantation. However, the effective implementation of HSPC gene therapy faces several major challenges. At present, a limited number of therapeutic retroviral vectors can be produced with good manufacturing practice (GMP) quality, resulting in the main bottleneck of the widespread use of this technology. In addition to the challenge of manufacturing a sufficient number of vectors, there is also a known risk of genetic toxicity associated with gene transfer using retroviral vectors, which is demonstrated by the development of malignant tumors caused by insertion mutagenesis (Hacein-Bey-Abina et al., Science, 302 (5644): 415-419 (2003); Hacein-Bey-Abina et al., N Engl J Med, 348 (3): 255-256 (2003); Ott et al., Nat Med, 12 (4): 401-409 (2006); and Stein et al., Nat Med, 16 (2): 198-204 (2010)). All these challenges have stimulated the development of non-viral means for genetic modification.

[0421] Most notably, gene editing has been proposed as a safer alternative to retroviral-mediated gene transfer, made possible by the development of engineered nucleases such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nucleases (Cornu et al., Nat Med, 23(4):415-423 (2017)), which are programmable nucleases that incorporate one or more RNA molecules to target specific sequences in DNA for cleavage by nuclease protein components. Among them, the Cas9 nuclease has been studied most thoroughly. This nuclease complexes with two RNA molecules, a guide RNA (crRNA) and a tracer RNA (tracrRNA), to recognize a homologous protospacer adjacent motif (PAM) site consisting of an NGG sequence, and then produces a blunt-ended double-strand break in the DNA. This break can be repaired by several cellular mechanisms, but the two most common ones are non-homologous end joining (NHEJ) and homology-directed repair (HDR) (Chang et al., Nature reviews Molecular cell biology, 18 (8): 495-506 (2017)), for the latter to occur, a complete template sequence homologous to the cleavage site must be present. Sister chromatids can be used as templates, but synthetic template molecules can also be provided in excess to improve HDR efficiency. Although the flanking regions of this template must significantly or completely match the flanking regions of the cleavage site, when HDR occurs, the new genetic code can be inserted into the genome, allowing the genome to be accurately edited or new DNA to be added to the genome, and for NHEJ, insertion and / or deletion (indel) is the most likely result (Chang et al., Nature reviews Molecular cell biology, 18 (8): 495-506 (2017)), recently, Cpf1 (or Cas12a) has also been shown to be useful in genome editing. This nuclease differs from Cas9 in that it recognizes a different protospacer adjacent motif (PAM) site (e.g., TTTN, where N can be A, C, G, or T), requires a single guide RNA, and enables staggered cleavage of DNA with 5' overhangs (Zetsche et al., Cell, 163(3):759-771 (2015)). It is speculated that the smaller size and staggered cleavage of Cpf1 enhance the ease of delivery and the potential for HDR when provided with a template oligonucleotide.

[0422] For maximum utility in HSPC gene therapy, a delivery platform containing designer-selected nucleases, with or without a DNA template, would be ideal, which is performed efficiently and reliably without cytotoxicity. The current clinical state of the art for this approach in HSPC requires electroporation of engineered nuclease components as mRNA or ribonucleoprotein (RNP) complexes. If HDR is preferred, the most effective method is electroporation followed by transduction with a non-integrating viral vector (Dever et al., Nature, 539(7629): 384-389 (2016)), or simultaneous electroporation of a specified concentration of an engineered nuclease component with a chemically modified single-stranded oligonucleotide (ssODN) template at a specified cell concentration (De Ravin et al., Sci Transl Med, 9(372)(2017)), electroporation is known to induce toxicity, and there is no means to control the number of cells that take up each component of the payload or the concentration of each component successfully delivered by electroporation (Lefesvre et al., BMC molecular biology, 3: 12-12 (2002)), and finally, when a non-integrating virus is used as a template, the system still relies on the availability of GMP-grade viral particles. Therefore, NP-based delivery is actively used to deliver gene editing components (Li et al., Human gene therapy, 26(7): 452-462 (2015)).

[0423] In this regard, lipid-based, polymer-based, and AuNPs have great potential for delivering gene editing components to cells (Finn et al., Cell Reports, 22(9):2227-2235 (2018); Lee et al., Nature Biomedical Engineering, 1(11):889-901 (2017); and Lee et al., Nature Biomedical Engineering, 2(7):497-507 (2018)). While polymer and lipid nanoparticles represent “encapsulation” and “entrapment” delivery vehicles, the unique surface loading of AuNPs facilitates precise modification and functionalization of different molecules such as RNA, DNA, and proteins (Rosi et al., Science, 312(5776):1027-1030 (2006)), because the surface area is known, controlled loading of payload components ensures uniformity of AuNP preparations, resulting in more predictable delivery (Ding et al., Molecular Therapy, 22(6):1075-1083 (2014)), and finally, AuNPs are considered to be relatively non-toxic compared to lipid and polymer nanocarriers (Pan et al., Small (Weinheim an der Bergstrasse, Germany), 3(11):1941-1949 (2007); Alkilany et al., Journal of Nanoparticles Research, 12 (7): 2313-2333 (2010); and Lewinski et al., Small (Weinheim an der Bergstrasse, Germany), 4 (1): 26-49 (2008)), which is critical for non-malignant dividing somatic cells such as HSPC. In fact, Lee et al. have demonstrated the effectiveness of polymer-encapsulated AuNP design in delivering CRISPR Cas9 and Cpf1 to non-dividing somatic tissues (such as muscle and brain) (Lee et al., Nature Biomedical Engineering, 1 (11): 889-901 (2017) and Lee et al., Nature Biomedical Engineering, 2 (7): 497-507 (2018)), but these carriers do not show efficacy in HSPC or accompanying oligonucleotide templates. In addition, the combination of polymer encapsulation and Au nano core greatly increases the overall NP size and changes the cytotoxicity characteristics of NP.

[0424] Simple Au-based gene-editing NPs (e.g., Au / CRISPR NPs) were designed, in which gene-editing components (guide RNA and nuclease) were conjugated layer-by-layer on the surface of AuNPs with or without single-stranded DNA template to support HDR (HDT), which did not require polymer encapsulation ( Figure 5C and 12A ).

[0425] The 19 nm AuNP cores were synthesized using the citrate reduction method (Turkevich et al., Discussions of the Faraday Society, 11(0):55-75 (1951)). The synthesized NPs were highly monodisperse with an observed polydispersity index (PDI) of 0.05 ( Fig. 12B and 12C ). Methods for the preparation and conjugation of the different layers can be found in Figure 5C In the first layer, CRISPR RNA (crRNA) for Cpf1 or Cas9 synthesized with an 18-nucleotide oligoethylene glycol (OEG) spacer and a terminal thiol linker (crRNA-18 spacer-SH) was attached to the surface of Au via semi-covalent Au-thiol interactions (sequence information can be found at Fig.34 ). Analysis of the published crystal structures of these Cas nucleases with crRNA and / or tracrRNA and double-stranded DNA showed that adding a spacer-thiol linker to crRNA had no effect on the recognition and nuclease activity of the guide segment (Yamano T et al., Cell, 165(4):949-962(2016) and Lee et al., eLife, 6:e25312(2017)), and the inclusion of an OEG spacer arm reduced the electrostatic repulsion between crRNA chains to increase the loading capacity on the AuNP surface. Fig. 12B As shown, the AuNP core with crRNA resulted in an NP size of 22nm and a PDI of 0.05. The nuclease protein was then attached to the 5' handle of the surface-loaded crRNA through the natural affinity of the nuclease for the 3D structure of crRNA. The nuclease connection increased the size of the NP to 40nm, with a PDI of 0.08 for Cpf1. This RNP-loaded AuNP was used as a basis for comparing nuclease activity in the absence of HDT. For HDT loading, the RNP-loaded AuNPs were further coated with branched low molecular weight (2000) polyethyleneimine (PEI) to prepare a substrate for electrostatic conjugation of HDT in the outermost layer. This "fully loaded" AuNP exhibited a size of 64nm and remained highly monodisperse, with an observed PDI of 0.17 ( Figures 12A-12CA homogeneous morphology without any aggregation was inferred from the transmission electron microscopy images, and fine localized surface plasmon resonance (LSPR) shifts were observed after each ligation step ( Fig. 12A , 12D NPs have a zeta potential change from -26 mV to +27 mV, with complete delamination ( Fig.12E ). This positive charge of the final NPs may prevent precipitation and aggregation over time, as these were not observed within a 48 h period after formulation.

[0426] This highly stable and monodisperse structure was attributed to the regulation of the weight / weight (w / w) ratio between AuNP and gene editing components. Analysis of different w / w ratios between AuNP and Cpf1 showed that lower ratios of Cpf1 could induce aggregation at an optimal w / w ratio of 0.6 ( Fig.13A , 13B At this ratio, the loading of Cpf1 was found to be 8.8 μg / mL. Compared with Cpf1, lower w / w ratios between AuNPs and HDT resulted in aggregation, with the optimal w / w ratio being 1 ( Fig. 13C , 13D ).

[0427] To determine the effect of this NP on primary HSPCs, HSPCs were isolated from leukapheresis products based on CD34 expression from healthy adult volunteers mobilized by granulocyte colony stimulating factor (G-CSF). Cells were cultured in support medium and the AuNP preparations were added to the cultures at a concentration of 10 μg / mL. Potential toxicity in CD34+ cells was analyzed by live-dead staining and trypan blue dye exclusion assays after 24 and 48 h of incubation with Au / CRISPR NPs ( Figures 15A-15C ), Au / CRISPRNP-treated samples showed more than 80% viability in both assays, with no difference between treated and untreated cells as determined by trypan blue.

[0428] Although HSPCs are known to be very difficult to transfect, confocal microscopy imaging showed good uptake and localization of gene editing components in the nucleus of primary HSPCs within 6 hours after treatment with Au / CRISPR NPs ( Figures 14A-14E Here, the cellular biodistribution of fluorescently labeled crRNA and HDT was tracked in z-series, and clear nuclear localization was observed in both cases ( Fig.14E ).

[0429] To test the utility of Au / CRISPR NP for gene editing, two different genomic loci were targeted that have shown therapeutic value in HSPCs: (1) the chemokine receptor 5 (CCR5) gene on chromosome 3, and (2) the gamma globin (γ-globin) gene promoter on chromosome 11. Disruption of CCR5 has been associated with resistance to human immunodeficiency virus (HIV) infection by eliminating viral binding and entry through the expressed CCR5 co-receptor (Lopalco et al., Viruses, 2(2):574-600 (2010)), and targeting this disruption in HSPCs renders future T cell progeny resistant to HIV infection. Alternatively, introduction of a specific deletion within the γ-globin promoter recapitulates a naturally occurring phenomenon known as hereditary persistent fetal hemoglobin (HPFH), which has been shown to be useful for treating hemoglobinopathies such as sickle cell disease and β-thalassemia (Akinsheye et al., Blood, 118(1):19 (2011)).

[0430] In silico off-target analysis of the CCR5 target using CasOFFinder software demonstrated that there are no homologous sites with less than 3 bp mismatches with Cpf1 in the human genome ( Figures 35A-35D ) (Bae et al., Bioinformatics, 30(10):1473-1475 (2014)), selecting a target site encoding both Cpf1 and Cas9 PAM sites accessible to a single guide RNA enabled direct comparison of these two CRISPR nucleases ( Fig. 7A , 7B ). However, before testing began, the HDT was optimized for Cpf1. Previous data suggested that cleavage of the non-target strand by the RuvC domain is a prerequisite for cleavage of the target strand by the Nuc domain (Yamano T et al., Cell, 165(4):949-962 (2016)), therefore, HDTs designed for both DNA targets and non-target strands were tested. This HDT consists of 40 bp homology arms flanking the Cpf1 cleavage site (1 7 bp downstream of the PAM), with an 8 bp Notl restriction enzyme cleavage site in the middle of each end to disrupt CCR5 expression and enable HDR analysis. Using Tracking Insertions and Deletions by Decomposition (TIDE), a total editing rate of 8.1% was observed for the non-target strand and a total editing rate of 7.8% was observed for the target strand, with 7.3% HDR when using an HDT designed for the non-target strand, compared to 5.4% HDR when using an HDT designed for the target strand ( Fig.21A ), these results were confirmed by T7EI and NotI restriction enzyme digestion assays ( Fig. 21B), and is closely related to the data previously published by Yamano T et al., Cell, 165(4):949-962 (2016).

[0431] The efficiency of HDR in primary HSPCs was then optimized by preparing Au / CRISPR-HDT-NPs at different concentrations (5 μg / mL-50 μg / mL) based on the amount of AuNP core suspended in molecular grade water. A concentration of 10 μg / mL demonstrated the highest total editing and HDR rates, with increasing concentrations demonstrating increased cytotoxicity and lower HDR rates ( Fig. 21C , 21D ).

[0432] Typically, in clinical procedures for ex vivo gene transfer, HSPCs are cultured on a recombinant fibronectin fragment. The final preparation for infusion into the patient consists of harvested HSPCs suspended in a pyrogen-free isotonic solution such as Plasma-Lyte containing 2% human serum albumin (HSA). To determine the effects of these agents, the cells were cultured in serum-free medium containing recombinant human growth factors on a 1:1 layer of HSA. Au / CRISPR-HDT NPs were tested for gene editing in the presence of either human A / B serum or combined human A / B serum. No changes in cytotoxicity were observed for any of the reagents ( Fig.22A ), but all agents reduced total editing and HDR rates ( Fig. 22B , 22C Therefore, for all subsequent experiments, HDTs designed for non-target DNA strands (included in the preparations) were added to HSPCs in culture at a concentration of 10 μg / mL in molecular grade water and HSPCs were cultured in the absence of or HSA-free serum-supportive medium.

[0433] It is assumed that staggered cleavage with 5' overhangs produced by Cpf1 is more conducive to HDR than blunt-end cleavage by Cas9 in HSPCs. To test this hypothesis, Au / CRISPR NPs targeting the CCR5 locus were prepared with and without HDT for Cpf1 and Cas9. For comparison, delivery was performed simultaneously with electroporation of each component at the same concentration. It is noteworthy that under any conditions, the guide RNA did not include additional chemical modifications such as 2'O-methyl ribonucleotides, 2-deoxy-2-fluoro-ribonucleotides, and thiophosphates (Yin et al., Nature Biotechnology, 35:1179 (2017)). TIDE analysis indicated a total editing range of 2% to 25%, with minimal significance ( Fig.23AHowever, increased incorporation of Notl restriction sites indicative of HDR was observed in HSPCs treated with Au / CRISPR NP-delivered Cpf1 or Cas9 compared with electroporation, with Cpf1 outperforming Cas9 ( Figures 23A-23C All cell viabilities were above 70% for all samples, but higher viabilities were observed in samples treated with AuNPs, and in particular significantly higher viabilities were observed when Cas9 was delivered via AuNPs rather than electroporation ( Fig.23D ). HSPC fitness in these samples was analyzed by colony forming cell (CFC) assay, and no differences in CFC potential or morphology were observed ( Fig.23E , 23F ). This standard CFC assay represents a shorter-term blood progenitor cell [Wognum B., Yuan N., Lai B., Miller CL (2013) Colony Forming Cell Assays for Human Hematopoietic Progenitor Cells. In: Helgason C., Miller C. (eds) Basic Cell Culture Protocols. Methods in Molecular Biology (Methods and Protocols), Vol. 946. Humana Press, Totowa, NJ], therefore, colonies from the original assay were re-plated as a measure of long-term repopulation capacity. No significant differences were observed in the number or type of secondary CFCs relative to the mock (untreated) control samples, but no pattern of higher CFC numbers was observed in the AuNP-treated samples relative to the electroporated samples ( Fig.24A , 24B ).

[0434] The same hypothesis was tested at the γ-globin promoter locus to confirm that Cpf1 is preferred for HDR. Here again, both Cpf1 and Cas9 PAM sequences were identified as having the same target cleavage site and no predicted off-target cleavage ( Fig. 8A , 8B ; Figures 35A-35D ), using HDT with a documented HPFH-associated 13-bp deletion inserted into this promoter that overlaps the repressor binding site (Akinsheye et al., Blood, 118(1):19 (2011)). Results obtained in primary HSPCs showed the same trend at this locus, with higher levels of HDR in Au / CRISPR NPs containing Cpf1 compared to NPs containing Cas9 ( Fig.25 ).

[0435] The next step was to determine whether NP treatment ex vivo impaired HSPC fitness after reinfusion. The best measure of HSPC fitness is the ability to reconstitute a myelosuppressed host. Therefore, primary human CD34+ HSPCs were treated ex vivo with Au / CRISPR-HDT-NPs and cultured at 10 6 Cells / mouse were infused into sublethally irradiated immunodeficient (NOD / SCIDγ- / -; NSG) mice. Mice were followed up for 22 weeks, with maximum engraftment observed at 8 weeks post-transplantation and stable engraftment established around 16 weeks post-transplantation ( Fig.27A Mouse body weight was monitored during the study and was stable over time ( Fig.28 Surprisingly, HSPCs treated with Au / CRISPR-HDT-NPs or AuNPs alone engrafted at higher levels than mock (untreated) cells, but with similar kinetics ( Fig.27B ). The different blood cell lineages were analyzed. B cell reconstitution peaked at 10 weeks after transplantation and then began to level off at 22 weeks ( Fig.27C Initial monocyte engraftment was high but decreased and stabilized over the first 8 weeks ( Fig.27D Low levels of T cells were observed until week 16, then increased in all study groups ( Fig.27E ). No significant differences in the proportions of B cells, monocytes, or T cells were observed relative to the ex vivo HSPC treatments administered.

[0436] Mice were sacrificed after 22 weeks, and bone marrow, spleen, thymus, and peripheral blood samples were removed. Flow cytometric analysis of autopsy samples showed that AuNP and Au / CRISPR-HDT-NP treated groups were associated with higher levels of engraftment compared with the mock group ( Figures 29A-29D Importantly, the frequency of multipotent CD34+ cells was higher in the bone marrow, spleen, and peripheral blood of AuNP-treated animals ( Fig.29A , 29B , 29D), and the frequency of cells expressing CD20 was higher in spleen, thymus, and peripheral blood ( Fig.29B , 29C , 29D). Human-specific CFC assays of bone marrow samples correlated well with engraftment results and showed that AuNP and Au / CRISPR-HDT-NP treated groups had significantly higher colony numbers compared with the mock treated group ( Fig.27F This was closely associated with the higher number of multipotent progenitor cells in these groups ( Figure 27G These results also correlated well with the CFC assay results observed in treated HSPC infusion products prior to transplantation, suggesting a positive effect of AuNP treatment in ex vivo cultured HSPCs ( Figures 30A-30B), the colony morphology of all treated samples is shown in Fig.31 middle.

[0437] In terms of gene editing, 9.8% total editing and 9.3% HDR were observed by TIDE analysis in HSPCs at the time of transplantation ( Fig.32A , 33 A steady level of total gene editing was observed in peripheral blood cells (5%), with a transient high of 17% observed at week 20 ( Fig.32B Interestingly, the level of NotI restriction enzyme incorporation remained below 1% at all time points ( Fig.32C ). Analysis of autopsy samples from different tissues showed that HDR was quite low in blood, bone marrow and spleen ( Fig.32D , 32E ).

[0438] Gene editing is a promising method for genetic screening to identify unknown genes, understand gene functions and correct defective genes in congenital or acquired genetic diseases (Xiong et al., Annual Review of Genomics and Human Genetics, 17 (1): 131-154 (2016)). Gene editing technology is rapidly developing from basic science to clinical application, but the current clinical technology for delivering gene editing components in HSPC requires electroporation, and AAV transduction may be required, which is much more complicated than retroviral-mediated gene transfer. Although all experience has been obtained from RNA, DNA and protein delivery, there is no effective and safe general simple method for delivering gene editing components, which indicates that different cell types and tissues may require different delivery strategies.

[0439] In this study, Au was used to develop a widely applicable gene editing delivery system. This multilayer NP can encapsulate all required gene editing components with or without a DNA repair template on a single AuNP core, with little effect on NP monodispersity. The strict characterization of each component loading step is crucial to the design. The best NP maintains a non-aggregated state and successfully infiltrates CD34+ hematopoietic cells that are difficult to transfect. Data from other cell types show that Au / CRISPR NP is internalized by endocytosis in vesicles, and then the vesicles rupture and are released into the cytoplasm. The proton sponge effect induced by PEI can promote escape from HSPC lysosomes (Benjaminsen et al., Molecular therapy: the journal of the American Society of Gene Therapy, 21 (1): 149-157 (2013)). Furthermore, PEI has been shown to play an active role in nuclear transport of NPs, which can promote payload delivery in addition to the nuclear localization signal on the nuclease protein (Reza et al., Nanotechnology, 28(2):025103 (2017)). The CCR5 and γ-globin promoter loci targeted here are quite unique, encoding PAM sites for Cpf1 and Cas9 with the same guide recognition site, enabling an unbiased comparison of these two nuclease platforms with this NP. Importantly, when Cpf1 nuclease was included in the NP, a 10 μg / mL Au / CRISPR NP concentration produced up to 17.6% total editing and 13.4% HDR at the CCR5 locus, and 12.1% total editing and 8.8% HDR at the γ-globin promoter locus. The total editing and HDR results were comparable or higher than electroporation-mediated delivery, suggesting that HSPC biology is more amenable to CRISPR gene editing when AuNPs are the delivery mode. Furthermore, higher levels of HDR observed with Cpf1 compared to Cas9 in NP suggest that staggered nuclease cleavage may favor HDR at least at these therapeutically relevant loci (Zetsche et al., Cell, 163(3):759-771 (2015) and Nakade et al., Bioengineered, 8(3):265-273 (2017)).

[0440] Colony assay results and xenograft data indicate that Au / CRISPR-HDT-NP treatment does not have any adverse effects on HSPC fitness after ex vivo treatment and suggests that it may even increase repopulation potential.

[0441] The evidence provided is that Au / gene editing NPs surprisingly yielded efficient and safe delivery of gene editing machinery to HSPCs. This study expands the available delivery toolkit for gene editing component delivery.

[0442] Materials. Synthesis and characterization of NPs. AuNPs were synthesized by a slightly modified Turkevich method (Turkevich et al., Discussions of the Faraday Society, 11(0):55-75(1951) and Shahbazi et al., Nanomedicine (London, England), 12(16):1961-1973(2017)). 0.25 mM chloroauric acid solution (Sigma-Aldrich, St. Louis, MO) was brought to boiling point and reduced by adding 3.33% sodium citrate solution (Sigma-Aldrich, St. Louis, MO) and vigorously stirred for 10 minutes under a reflux system. The synthesized NPs were washed three times by centrifugation at 17000 for 15 minutes and redispersed in ultrapure water (Invitrogen, Carlsbad, CA).

[0443] All oligonucleotides used in this study were purchased from Integrated DNA Technologies (IDT, Coralville, IA). Cas9 and Cpf1 enzymes were purchased from Aldevron, LLC (Fargo, ND). crRNAs modified with 18 oligoethylene glycol (OEG) spacers-thiol at the 3' end for AsCpf1 and the 5' end for SpCas9 were used (sequence information can be found at Fig.34Found in ). The crRNA and tracrRNA duplexes (gRNA) for Cas9 nuclease were prepared by mixing them in duplex buffer at equimolar concentrations and incubating at 95 ° C for 5 minutes and cooling on the work surface. 10 μg / mL concentration of AuNP was added to the crRNA or gRNA solution at an AuNP / crRNA w / w ratio of 0.5. Citrate buffer (pH3.0) was added to 10mM, and the resulting solution was mixed for 5 minutes. The prepared AuNP / crRNA nanoconjugate was centrifuged and redispersed in 154mM sodium chloride (NaCl) (Sigma-Aldrich, St.Louis, MO). Then, nuclease was added at an AuNP / Cpf1 or AuNP / Cas9 w / w ratio of 0.6, and mixed by pipetting up and down and incubating for 15 minutes. Then, NP was centrifuged at 16000g for 15 minutes and redispersed in NaCl solution. 2000 MW polyethyleneimine (PEI) (Polysciences, Philadelphia, PA) was added at a concentration of 0.005%, mixed thoroughly, and incubated for 10 minutes before centrifuging the NPs at 15000 g for 15 minutes and redispersing them in NaCl solution. In the final step, HDT was added at a AuNP / HDT w / w ratio of 2 and incubated for 10 minutes before centrifuging the NPs and redispersing them in NaCl solution.

[0444] The size and shape of the prepared NPs were characterized by transmission electron microscopy (TEM) (JEOL JEM 1400, Akishima, Tokyo, JP), and the samples were first negatively stained by glow discharge carbon coated grids using the PELCO easiGlow Glow Discharge system (Ted Pella Inc., Redding, CA). A 2 μL volume of sample was dropped onto the grid and blotted dry after 30 seconds, washed and stained in 0.75% uranyl formate solution (Polysciences, Philadelphia, PA). Finally, the grid was dried overnight in a desiccator and imaged by TEM (Booth et al., JoVE (58): 3227 (2011)).

[0445] The hydrodynamic size and polydispersity index of NPs were characterized by a Zetasizer Nano S device (Malvern, UK). The measurements were performed in triplicate and the results are reported as mean ± SD. The measurements were performed using a small volume disposable cuvette (ZEN0040) (Malvern, UK).

[0446] The zeta potential of the NPs was characterized by using a Zetasizer Nano ZS (Malvern, UK). The measurements were performed using a disposable folded capillary zeta cell (Malvern, UK) and the results are reported as mean ± SD.

[0447] Furthermore, the layer-by-layer conjugation of CRISPR components was characterized by measuring the shift of the localized surface plasmon resonance (LSPR) of AuNPs using a nanodrop device (Thermo Fisher Scientific, Waltham, MA).

[0448] Isolation and culture of CD34+ cells. Primary human CD34+ cells were isolated from healthy donors mobilized with granulocyte colony stimulating factor (G-CSF; filgrastim, Amgen, Thousand Oaks, CA). Complete leukapheresis products were obtained and cultured in CliniMACS using a previously published protocol (Adair et al., Nat Commun, 7:13173 (2016)). TM CD34-expressing cells were purified by immunomagnetic bead-based separation on a Prodigy device and cultured in StemSpan serum-free expansion medium version II (SFEMII; Stem Cell Technologies) or Iscove's modified Dulbecco's medium (IMDM; Invitrogen Life Sciences, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Gibco, Waltham, MA) and 100 ng / mL each of recombinant human stem cell factor (SCF), Flt-3 ligand (Flt3), and thrombopoietin (TPO) from Cellgenix (Freiburg, Germany). Culture conditions were 37°C, 85% relative humidity, 5% CO2, and normoxia.

[0449] In vitro gene editing studies. CD34+ cells were thawed and pre-stimulated overnight in SFEMII medium containing SCF, Flt3, and TPO. Afterwards, cells were incubated at 1x10 6 Cells were seeded in 96-well plates at 10 μg / mL and treated with Au / CRISPR NPs at a concentration of 10 μg / mL. All in vitro experiments were performed in triplicate. After 48 h of incubation, cells were washed with Dulbecco's phosphate-buffered saline (D-PBS) (Gibco, Waltham, MA) and harvested for gDNA extraction and gene editing analysis.

[0450] Electroporation of CRISPR components was also performed for comparison. To this end, 49pmol crRNA or gRNA was mixed with the same amount of Cpf1 or Cas9 nuclease (8.5pmol) and incubated for 15 minutes. The cells were dispersed in electroporation buffer and mixed with ribonucleoprotein (RNP) complex. The mixture was added to a 1mm electroporation cuvette and electroporated at 250V and 5ms pulse duration using a BTX electroporation device (BTX, Holliston, MA). Thereafter, the cells were placed in culture and washed after 24 hours, then incubated for another 24 hours. After incubation for 48 hours, the cells were washed with D-PBS and harvested for gDNA extraction and gene editing analysis.

[0451] Cell viability analysis. Cell viability after treatment with Au / CRISPR NPs and electroporation was analyzed at different time points using a Countess II FL automated cell counter (ThermoFisherScientific, Waltham, MA). 10 μL of trypan blue dye (0.4%) (Invitrogen) was mixed with 10 μL of cell suspension and 10 μL of the mixture was applied to a disposable cell counting chamber slide and inserted into the device. The cell viability percentage of each sample was recorded and reported as mean ± SD.

[0452] To confirm the results, we also used Cell viability was analyzed using the ELISA kit (Invitrogen, Carlsbad, CA). Cells were washed with D-PBS and pelleted by centrifugation. Then, aliquots of the cell suspension were transferred to coverslips. The cells were allowed to settle to the surface of the glass coverslips in a covered 35 mm dish at 37°C. Calcein AM (2 μM) and ethidium homodimer-1 (EthD-1) (4 μM) working solutions were prepared and 150 μL of the combined The assay reagent was added to the surface of a 22 mm square cover glass so that all cells were covered with the solution. The cells were incubated in a covered culture dish for 30 minutes at room temperature. After incubation, 10 μL of D-PBS was added to a clean microscope slide, and the cover glass was inverted and mounted on a microscope slide. The labeled cells were imaged using the excitation and emission values ​​of 494 / 517 nm (for calcein AM) and 528 / 617 nm (for EthD-1) under a fluorescence microscope (Nikon TiLive, Japan). Live and dead cells were counted using cellomics vHSC software (v1.6.3.0, Thermo Fisher Scientific, Waltham, MA). Images were processed using Imaged software (V 1.5i, National Institutes of Health, Rockville, MD).

[0453] Colony forming cell (CFC) assay. For CFC assay, cells were plated in methylcellulose containing recombinant human growth factor (H4230: Stem Cell Technologies, Vancouver, CA) and incubated for a period of 14 days according to the manufacturer's instructions. The resulting colonies were counted and morphologically scored on a stereomicroscope (ZEISS Stemi 508, Germany) to determine the number of cells forming colonies per 100,000 plated cells.

[0454] Genome editing was detected by T7 endonuclease I. To analyze the total gene editing percentage, use Genomic DNA was extracted and PCR amplified using the GENE DN A mini kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol.

[0455] The genomic region (755 bp) flanking the CRISPR target site was PCR amplified (sequence information can be found at Fig.34 ) and use according to the manufacturer's protocol The product was purified using a PCR purification kit. A total of 200 ng of purified PCR product was mixed with 2 μL 10x NEBuffer2 (New England BioLabs, Ipswich, MA) and ultrapure water to a final volume of 19 μL and subjected to a reannealing process to enable heteroduplex formation: 95°C for 5 minutes, -2°C / s from 95°C to 85°C, -0.1°C / s from 85°C to 25°C, and held at 4°C. After reannealing, the product was treated with 1 μL T7EI nuclease (New England BioLabs, Ipswich, MA) and incubated at 37°C for 15 minutes. After incubation, the product was purified by The digested products were purified by PCR purification kit and analyzed on 2% agarose gel. The gel was imaged with Gel Doc gel imaging system (Bio-Rad, Hercules, CA). Quantification was performed based on relative band intensity. The percentage of indels was determined by the formula, % gene modification = 100x (1-(1-cleavage fraction) 1 / 2).

[0456] The genomic region flanking the CRISPR target site (755 bp) was PCR amplified and used according to the manufacturer's protocol. Purify the product using a PCR purification kit. Mix a total of 1000 ng of purified PCR product with 5 μL Buffer (New England BioLabs, Ipswich, MA), 1 μL Notl enzyme (New England BioLabs, Ipswich, MA) and ultrapure water were mixed to a final volume of 50 μL. After incubation at 37°C for 15 minutes, The digested products were purified by PCR purification kit and analyzed on 2% agarose gel. The gel was imaged with Gel Doc gel imaging system (Bio-Rad, Hercules, CA). Quantification was performed based on relative band intensity. The percentage of gene insertion was determined by the formula, % gene modification = 100x (1-(1-cleavage fraction) 1 / 2).

[0457] Genome editing was detected by TIDE assay. The genomic region (755 bp) flanking the CRISPR target site was PCR amplified (sequence information can be found at Fig.34 ) and use according to the manufacturer's protocol Purify the product with a PCR purification kit. Sanger sequencing was performed by mixing the final volume of 10 μL with ultrapure water and Terminator (Thermo Fisher Scientific, Waltham, MA). After cycle sequencing, samples were analyzed by 3730x1 DNA analyzer (Applied Biosystems, Foster City, CA). The resulting sequences were run on TIDE software (https: / / tide.nki.nl / ), and the results were reported as percentage of gene modification (Brinkman et al., Nucleic Acids Research, 42(22): e168-e168(2014)).

[0458] MiSeq analysis. The first PCR was performed on the genomic region (755 bp) flanking the CRISPR target site (sequence information can be found at Fig.34 ). and use according to the manufacturer's protocol. The product was purified using a PCR purification kit. A second PCR was performed using primers with Miseq adapter sequences on the genomic region (157 bp) flanking the CRISPR target site and using The product was purified using a PCR purification kit and specific bands were checked by running 5 μL of the sample on a 2% agarose gel. The DNA was then indexed using the Nextera Indexing Kit (96 indexes) (Illumina, San Diego, CA) for 8 cycles. PCR purification kit was used to purify the product. Finally, the prepared library was diluted to 4 nM, merged and analyzed by Illumina HiSeq 2500 (Illumina, San Diego, CA). Sequencing reads were analyzed using an internal bioinformatics pipeline. Paired high-throughput sequencing reads (Miseq) were combined with PAIR [PMID 24142950]. The combined reads were then filtered using a custom python script. Reads without complete primer sequences were discarded. Primer sequences were trimmed from the reads, and then identical sequences were combined. The Needleman-Wunsch aligner from the emboss suite was used to align sequence reads with reference amplicons [PMID 5420325, Kruskal, JB, (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley]. The options used for this aligner are: -gapopen 10.0, -gapextend 0.5, and -aformat3 sam. A custom python script then reads the Concise Specific Gap Alignment Report (CIGAR) string from the sequence alignment map (SAM) output and uses this information to identify and quantify insertions and deletions. Each aligned sequence is also compared to the reference amplicon to identify substitution mutations. Any mutation found in only one read is removed from the analysis. A table containing the mutant sequence, read count, and frequency for each mutation is then output for further analysis. In each sequencing run, a control sample consisting of electroporated cells from the same animal was determined for the average frequency of the mutation class (insertion, deletion, substitution, insertion and substitution, etc.) before transplantation and was used to perform a one-tailed binomial t-test for each mutation from the corresponding mutation class. Mutations from experimental samples were retained if a p-value < 0.05 was demonstrated. All custom scripts are available upon request.

[0459] In vivo implantation studies in NSG mice. All experiments involving animals were performed in accordance with the Office of Laboratory Animal Welfare (OLAW) Public Health Safety (PHS) policy, the United States Department of Agriculture (USDA) Animal Welfare Act and regulations, the Guide for the Care and Use of Laboratory Animals, and IACUC protocol 1864 in accordance with regulatory agency guidelines.

[0460] NOD. Cg-Prkdcscidll2rgtm1Wjl / Szj (NOD SCIDγ- / -; NSG) mice were obtained from The Jackson Laboratory and housed in-house under pathogen-free conditions. Adult mice (8-12 weeks old) received whole-body irradiation of 175 cGy from a cesium irradiator and 3-4 hours later received a single intrahepatic injection of 1×10 resuspended in 30 μL phosphate-buffered saline (PBS; Invitrogen Life Sciences) containing 1% heparin (APP Pharmaceuticals). 6 Primary human CD34+ hematopoietic cells. Four weeks after implantation, blood was collected by retroorbital puncture to determine the level of human blood cells by flow cytometry. Blood was collected every two weeks during follow-up. As previously reported (Haworth et al., Mol Ther Methods Clin Dev, 6: 17-30 (2017)), leukocytes were separated and stained with anti-human CD45 antibody (clone 2D1), anti-human CD3 antibody (clone UCHT1), anti-human CD4 antibody (clone RPA-T4), anti-human CD20 antibody (clone 2H7) and anti-human CD14 antibody (clone M5E2) antibody (all from BD Biosciences, San Jose, CA), and stained cells were obtained on FACS Canto II (BD Biosciences, San Jose, CA), and analyzed using FlowJo software v10.1 (Tree Star).

[0461] Confocal microscopy imaging. To track intracellular biodistribution, Cpf1 crRNA and HDT (IDT, Coralville, IA) were fluorescently labeled at the 5' end with Alexa 488 and Alexa 660 fluorophores, respectively, to prepare Au / CRISPR NPs and incubate with cells for 6 h. At the end of the incubation, cells were washed and dispersed in FluoroBrite in a FluoroDish. TM Two drops of NucBlue were added to the cells. TM Live ReadyProbes TMReagent (Ex / Em 360 / 460nm) (Invitrogen, Carlsbad, CA) and incubated at room temperature for 30 minutes. Finally, cells were imaged on a Zeiss LSM 780 Confocal and Multi-Photon with an Airyscan microscope (Zeiss, Germany). Images were analyzed using ZEN Lite software (Zeiss, Germany). Imaging was performed using a 60x objective lens after background adjustment.

[0462] Statistical analysis. All data were reported as mean ± SD and statistically analyzed using paired Student's t test and GraphPad Prism software (Windows version 7.03) (GraphPad Software, USA). p values ​​< 0.05 were considered statistically significant.

[0463] Example 3. In vitro targeting efficiency. The purpose of this example was to show that NPs can target specific blood cell types (HSPCs or T cells) in a mixed cell population (unprocessed blood or bone marrow product).

[0464] Current clinical gene therapy in blood cells requires purification of target immune cells (e.g., HSPCs or T cells) from other blood cell types. NPs that can specifically bind to and deliver gene editors to immune cells without purification will greatly simplify the current gene therapy manufacturing process because it will not require ex vivo purification and culturing of cells for patient-specific cell therapy. In addition, this will accelerate the potential for in vivo delivery of gene editing to blood cells, which represents the most portable gene therapy strategy overall. This highly simplified manufacturing strategy is called the "minimum manipulation" approach.

[0465] The cell types tested in this embodiment include: 1) primary human HSPC (CD34+ cells and / or CD34+ / CD45RA- / CD90+ cells), and 2) primary human T cells (CD3+ and CD4+ cells). Clinically relevant sources of HSPC include bone marrow, peripheral blood mobilized by granulocyte colony stimulating factor (GCSF), and peripheral blood mobilized by AMD3100 (plerixafor). Clinically relevant sources of T cells include whole peripheral blood.

[0466] The genetic loci to be edited include: 1) the γ-globin promoter in HSPCs, which is associated with hemoglobinopathies such as sickle cell disease; and 2) CCR5 in T cells, which is associated with HIV infection.

[0467] Targeting molecules to be tested in HSPCs include: a) antibodies that bind CD34, CD90, or CD133 (tested alone and in combinations of 2); b) aptamers that bind CD133 (tested alone and in combination with antibodies or ligands); and c) ligands: human chorionic gonadotropin (HCG) and SR1 (Stem Regenin 1). Targeting molecules to be tested in T cells include: a) antibodies that bind CD3, CD4 (tested alone and in combination); and b) aptamers that bind CD3 (tested alone and in combination with antibodies). The chemistry required to add each of these molecule types to existing NPs will utilize amine-thiol or thiol-thiol crosslinkers with various PEG spacers.

[0468] Unmanipulated blood cell products from healthy donors will be divided into aliquots, one for each targeting molecule or combination or set thereof. Each targeting molecule will be tested as a surface display material for NPs. To track uptake, the guide RNA (innermost layer) is labeled with a far-red fluorescent dye. Target and non-target cell populations are tracked with fluorescently labeled antibodies using different wavelength fluorophores below far-red light. For each of the above blood cell sources, the experiment will be repeated for a minimum of 6 and a maximum of 10 unique donors (biological replicates).

[0469] Confocal microscopy and flow cytometry will be used to assess the uptake of NPs by target and non-target cells. For both assays, indications for selecting targeted molecules, cell types, and / or blood products for further testing may include: (i) a minimum of 50% and a maximum of 100% of target cells display a red fluorescent phenotype, and (ii) a minimum of 0% and a maximum of 20% of non-target cells display a red fluorescent phenotype. Criteria for selecting targeted molecules, cell types, and / or blood products for further testing may include: (i) for at least one experimental group in one clinically relevant cell type, an average of ≥50% of target cells (HSPCs or T cells) red fluorescence was observed in the donor, and (ii) for any other non-target cell type, ≤20% red fluorescence was observed in the donor.

[0470] Criteria for eliminating targeted molecules, cell types, and / or blood products from further testing may include: (i) <50% target cell uptake observed under all experimental conditions tested, or (ii) >20% non-target cell uptake.

[0471] This study will determine which of the tested targeting molecules best selectively associates NPs with desired cellular phenotypes in unmanipulated, clinically relevant blood cell products.

[0472] Example 4. Preclinical Evaluation of Minimally Manipulated Cell Products in Vitro This example was to demonstrate that the disclosed NPs are a clinically feasible strategy to achieve "minimal manipulation" of blood cell products for gene therapy, thereby eliminating the need for ex vivo purification and target cell culture.

[0473] For the clinical transformation of the targeted NPs, the feasibility of manufacturing minimally manipulated blood cell products at clinical scale that meet current standards for reinfusion into human patients (see Table 3) will be demonstrated. The AuNP-based gene editing delivery system disclosed herein with and without targeting molecules (identified from Example 3) will be tested in unmanipulated human donor blood products at clinical scale to demonstrate the feasibility of scale-up. Such feasibility data is critical to establishing a translational manufacturing method for patient-specific cell therapies that does not include purification, culture, electroporation, or engineered viruses.

[0474] Specific blood products and cell types associated with indications or criteria for further testing (from Example 3) will be the target of this example. When more than one cell type and blood product meets the criteria for further testing, the cell type and blood product with the highest performance (i.e., highest level of gene editing and best targeting potential) will be further tested first, followed by lower performing candidates.

[0475] Clinically relevant sources of HSPCs and T cells are as described in Example 3: (i) for HSPCs, bone marrow, GCSF-mobilized peripheral blood, and AMD3100 (plerixafor)-mobilized peripheral blood; and (ii) for T cells, whole peripheral blood.

[0476] The genetic loci to be edited are as described in Example 3: 1) γ-globin promoter in HSPCs; and 2) CCR5 in T cells.

[0477] Blood / bone marrow products from at least three individual donors will be collected. Each product from each donor will be divided into three equal aliquots: one for no treatment (mock control), one for treatment with the (non-targeted) AuNP-based gene editing delivery system of the present disclosure, and one for treatment with the AuNP-based gene editing delivery system of the present disclosure + selected targeting molecules.

[0478] The assays to be used in this embodiment include: fluorescence-assisted cell sorting (FACS) or immunomagnetic bead-based sorting, gene editing analysis, trace element analysis by inductively coupled plasma mass spectrometry (ICP-MS), viability assays, and release tests (i.e., suitability for reinfusion testing). For cell sorting by FACS or immunomagnetic beads, the minimum purity of the target cell pool required to fully evaluate all other parameters is ≥90%, and the maximum purity is 100%. There is no threshold requirement for the purity of the non-target (negative) portion. For gene editing analysis, the minimum threshold for the target cell phenotype is 20% total gene editing, and the maximum is 50% gene editing; the minimum threshold for the non-target cell phenotype is 0% gene editing, and the maximum is 20% gene editing. The product must meet the standard release criteria for reinfusion of autologous gene-modified cell products (see Table 3 below). Trace element analysis will be performed on the final product formulated for infusion, with the sole purpose of understanding the amount of Au present. There is no minimum threshold, and the maximum value cannot exceed the total mass added by the initial treatment (the maximum value is 10 μg / mL starting cell product). This data will be used to evaluate in vivo biodistribution and clearance in Example 5 when the selection criteria discussed below are met.

[0479] Criteria for selecting NPs for further testing may include: (i) an average of ≥20% total gene editing observed only in target cells of the donor, and (ii) ≥70% cell viability and meeting all other release criteria.

[0480] This example can demonstrate that the selected NPs are suitable for minimal manipulation approaches to human blood cell products, or which cell types or blood product components (serum, macrophages, etc.) are the greatest barriers to success.

[0481] Table 3. Standard release criteria for autologous genetically modified cell products to be reinfused.

[0482]

[0483]

[0484] Final release sterility testing performed includes sterilization in a controlled cleanroom in accordance with USP <71> Guidelines for testing bacteria, fungi and yeast after 14 days of incubation.

[0485] ε According to USP <71> Guidelines for testing by institutional quality control using the Limulus amebocyte lysate (LAL) test.

[0486] Example 5. Preclinical Evaluation of Minimally Manipulated Human Cell Products in Vivo This example demonstrates the preclinical safety and feasibility of a minimally manipulated human blood cell product in an immunodeficient mouse model.

[0487] The established model for demonstrating the safety and efficacy of genetically modified human blood cells is xenotransplantation. In this model, human blood cells are transplanted into irradiated immunodeficient mice. This model allows cells to be transplanted from one human donor into multiple individual mice. The parameters that can be studied in this model include blood cell performance, toxicity, biodistribution and removal in animals. Importantly, it is expected that some AuNPs can still be present in the least manipulated blood cell products when reinfused, and this study can contribute to understanding the physiological impact of NP administration. This information is important for the clinical transformation of the method, and will also provide information for direct in vivo administration studies. In this embodiment, the least manipulated human blood cell products (from Example 4) selected for further study are injected into sublethally irradiated immunodeficient mice to monitor cell performance (implantation), as well as biodistribution and removal of any residual NPs infused together with the blood cell products. This can be considered as an experiment of "risk reduction" of the disclosed technology.

[0488] Specific blood products and cell types selected for further study from Example 3 will be the targets of these studies.

[0489] Clinically relevant sources of HSPCs and T cells are as described in Examples 3 and 4: (i) for HSPCs, bone marrow, GCSF-mobilized peripheral blood, and AMD3100 (plerixafor)-mobilized peripheral blood; and (ii) for T cells, whole peripheral blood.

[0490] The genetic loci to be edited are as described in Examples 3 and 4: 1) γ-globin promoter in HSPCs; and 2) CCR5 in T cells.

[0491] Minimally manipulated blood / marrow products from three individual donors in Example 4 will be infused into immunodeficient mice within 12-24 hours after sublethal whole-body irradiation. Human cell engraftment, as well as engraftment of gene-edited cells and the general health of the animals will be monitored over time after transplantation. Imaging, urine, and feces can be obtained from these mice after infusion to determine the biodistribution and clearance of NPs that may be present in the infused product.

[0492] Assays and experiments that will be performed in the study include: visual monitoring of the health of infused mice (grooming, weight, and activity level); hematological recovery after transplantation; engraftment and persistence of gene-edited cells; trace element analysis of the infused product by ICP-MS; and analysis of urine and feces by ICP-MS 72 hours after infusion to determine if all NPs have been cleared (mass balance). If bioaccumulation is indicated, micro-computed tomography (CT) imaging of live mice can be performed to assess the location of accumulation. If accumulation is too low to be observed with micro-CT, necropsy and additional trace element analysis by ICP-MS can be performed to determine the site of bioaccumulation. Micro-CT, necropsy, and / or trace element analysis can be combined with histopathology to assess potential toxicity. The reading thresholds for these various assays are described in the following paragraphs.

[0493] Engraftment and persistence. Flow cytometry can be used to assess the levels of cells expressing human CD45 in the blood, bone marrow, and spleen. The minimum threshold is 0%, and the maximum threshold is 100%.

[0494] Gene Editing Analysis. The minimum threshold is 5% in human cells and the maximum threshold is 100%. It is expected that sufficient NPs will remain in the preparation to edit mouse cells; however, as described below, the assay will evaluate whether gene editing is detected in mouse CD45-expressing cells or any tissue that shows bioaccumulation.

[0495] Health monitoring. Each mouse will be evaluated for pain and distress (minimum PD1, maximum PD4) and body condition (minimum BC1, maximum BC5) before NP administration, then daily for 3 days after NP administration, and weekly thereafter. Scoring is based on the publication of Burkholder et al. Health Evaluation of Experimental Laboratory Mice. Current Protocols in Mouse Biology, 2012; 2: 145-165. Any adverse reactions will be recorded and summarized.

[0496] Trace element analysis. The minimum threshold in urine / feces is 0 within 72 hours, and the maximum threshold cannot exceed the total mass injected. The minimum threshold in tissues is 0, and the maximum threshold cannot exceed the total mass injected.

[0497] Micro-CT imaging. The minimum threshold is no contrast enhancement, and the maximum threshold is to be determined.

[0498] Histopathology. The assay will assess significant organ toxicity relative to untreated controls from all donors. The minimum threshold is no toxicity and the maximum threshold is graded using published adverse event criteria for each target organ.

[0499] The studies described in this example will establish the preclinical in vivo safety and efficacy of a minimally manipulated human blood product.

[0500] (XIV) End of paragraph. The disclosed nucleic acid sequences are shown using standard letter abbreviations for nucleotide bases, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but it is understood that the complementary strand is included.

[0501] Variants of the proteins and / or nucleic acid sequences disclosed herein may also be used. Variants include sequences having at least 70% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the proteins and nucleic acid sequences described or disclosed herein, wherein the variants exhibit substantially similar or improved biological functions.

[0502] "% sequence identity" refers to the relationship between two or more sequences as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein and nucleic acid sequences, as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, ed.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., ed.) Oxford University Press, NY (1992). Preferred methods for determining identity are designed to obtain the best match between tested sequences. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignment and identity percentage calculation can be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignments of sequences can also be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (gap penalty=10, gap length penalty=10).Related programs also include the GCG program package (Wisconsin package version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program in conjunction with the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor: Suhai, Sandor. Publisher: Plenum, New York, NY. Within the context of the present disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the referenced program. "Default values" shall mean any set of values ​​or parameters that are initially loaded with the software when it is initially initialized.

[0503] In a specific embodiment, the variant protein comprises a conservative amino acid substitution. In a specific embodiment, a conservative amino acid substitution cannot substantially change the structural characteristics of the reference sequence (e.g., the replacement amino acid should not tend to interrupt the helix present in the reference sequence, or destroy other types of secondary structures that characterize the reference sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature, 354:105 (1991).

[0504] In a specific embodiment, a "conservative substitution" relates to a substitution found in one of the following conservative substitution groups: Group 1: alanine (Ala), glycine (Gly), serine (Ser), threonine (Thr); Group 2: aspartic acid (Asp), glutamic acid (Glu); Group 3: asparagine (Asn), glutamine (Gin); Group 4: arginine (Arg), lysine (Lys), histidine (His); Group 5: isoleucine (lie), leucine (Leu), methionine (Met), valine (Vai); and Group 6: phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp).

[0505] In addition, amino acids can be grouped into conservative substituents by similar function or chemical structure or composition (e.g., acidic, basic, aliphatic, aromatic, sulfur-containing). For example, for substitution purposes, an aliphatic group can include Gly, Ala, Vai, Leu, and Ile. Other groups containing amino acids that are considered to be conservative substitutes for each other include: sulfur-containing: Met and cysteine ​​(Cys); acidic: Asp, Glu, Asn, and Gin; small aliphatic nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic nonpolar residues: Met, Leu, Ile, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information is found in Creighton (1984) Proteins, WH Freeman and Company.

[0506] In certain embodiments, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of an antibody and its target marker. Unless otherwise indicated, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (i.e., antibody and target marker). The affinity of an antibody for its target marker can be generally expressed by a dissociation constant (Kd) or an association constant (K A Affinity can be measured by common methods known in the art.

[0507] As will be appreciated by those of ordinary skill in the art, there are many commercially available antibodies and targeting ligands that bind to the cellular markers described herein.

[0508] In certain embodiments, binding affinity can be assessed under relevant in vitro conditions, such as buffered saline at near physiological pH (7.4) at room temperature or 37°C.

[0509] In certain embodiments, "binding" refers to an antibody that binds to -8 M or less, in a specific embodiment 10 -5 M to 10 -13 M. In a specific embodiment 10 -5 M to 10 -10 M. In a specific embodiment 10 -5 M to 10 -7 M. In a specific embodiment 10 -8 M to 10 -13 M, or in a specific embodiment 10 -9 M to 10 -13 The term may further be used to indicate that an antibody does not bind to other biomolecules present (e.g., it binds to its target marker with a dissociation constant (D) of 10 -4 M or more, in a specific embodiment 10 -4 M to 1 M dissociation constant (KD) for binding to other biomolecules).

[0510] In certain embodiments, "binding" means that the antibody binds to 7 M -1 or higher affinity constant (i.e., association constant, K A ), in a specific embodiment 10 5 M -1 Up to 10 13 M -1 In a specific embodiment 10 5 M -1 Up to 10 10 M -1 In a specific embodiment 10 5 M -1 Up to 10 8 M_ 1 In a specific embodiment 10 7 M -1 Up to 10 13 M -1 , or in a specific embodiment 10 7 M -1 Up to 10 8 M -1 The term can further be used to indicate that the antibody does not bind to other biomolecules present (e.g., it binds to a target marker at 10 4 M -1 or less, in a specific embodiment 10 4 M -1 Up to 1M -1 The association constant (K A ) binds to other biomolecules).

[0511] As noted, specific embodiments may utilize variants of the targeting ligand binding domain. Variants of the targeting ligand binding domain may include variants having one or more conservative amino acid substitutions or one or more non-conservative substitutions that do not adversely affect the binding of the antibody to the targeted epitope.

[0512] In certain embodiments, when compared to antibodies generated and characterized according to the methods disclosed herein, V L The region may include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the above changes. Insertions, deletions, or substitutions may be in V L Anywhere in the region, including at the amino or carboxyl termini or both termini of this region, provided that each CDR includes zero alterations or at most one, two or three alterations, and provided that the modified V L Antibodies targeting the region may still specifically bind to the targeted epitope with similar affinity as the reference antibody.

[0513] In certain embodiments, when compared to antibodies generated and characterized according to the methods disclosed herein, V H The region may be derived from or based on the disclosed V H , and may include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above changes. Insertions, deletions or substitutions may be in V H Anywhere in the region, including at the amino or carboxyl termini or both termini of this region, provided that each CDR includes zero alterations or at most one, two or three alterations, and provided that the modified V H Antibodies targeting the region can still specifically bind to their target epitope with similar affinity as the reference antibody.

[0514] Those of ordinary skill in the art understand the references to CD34, CD45RA, CD90, CD117, CD123, CD133, CD164 and other CDs described herein. For other readers, CD (cluster of differentiation) antigens are proteins expressed on the surface of cells that can be detected by specific antibodies. CD34 is a highly glycosylated type I transmembrane protein expressed on 1-4% of bone marrow cells. CD45RA is related to type III fibronectin, has a molecular weight of 205-220 kDa, and is expressed on B cells, naive T cells and monocytes. CD90 is a GPI-cell anchor molecule found on human prothymocytes. CD117 is a c-kit ligand receptor found on 1-4% of bone marrow stem cells. CD123A is related to the cytokine receptor superfamily and the fibronectin type III superfamily, has a molecular weight of 70 kDa, and is expressed on bone marrow stem cells granulocytes, monocytes and megakaryocytes. CD133 is a five-transmembrane glycoprotein expressed on primitive hematopoietic progenitor cells and other stem cells. CD164 is a type I integrin transmembrane sialomucin expressed by human hematopoietic progenitor cells and bone marrow stromal cells.

[0515] Unless otherwise indicated, the practice of the present disclosure may employ conventional techniques of immunology, molecular biology, microbiology, cell biology, and recombinant DNA. These methods are described in the following publications. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition (1989); FM Ausubel et al., Current Protocols in Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991); MacPherson et al., ed. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, A Laboratory Manual, (1988); and RI Freshney, ed. Animal Cell Culture (1987).

[0516] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein may include, consist essentially of, or consist of the following: its specifically stated elements, steps, ingredients, or components. Therefore, the term "include" or "including" should be understood as a narrative: "comprise, consist of, or consist essentially of". The transitional term "comprise" or "comprises" means including, but not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in larger amounts. The transitional phrase "consisting of" excludes any unspecified elements, steps, ingredients, or components. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the specified elements, steps, ingredients, or components and those that do not substantially affect the embodiment. The substantial impact will cause a statistically significant reduction in the ability of the desired cell type in the ex vivo blood cell product that has been subjected to minimal manipulation to selectively genetically modify.

[0517] Unless otherwise indicated, all numbers used in the specification and claims expressing the amounts of ingredients, properties such as molecular weight, reaction conditions, etc. should be understood as being modified in all cases by the term "about". Therefore, unless indicated to the contrary, the numerical parameters listed in the specification and the appended claims are approximate values, which may vary depending on the desired properties sought to be obtained by the present invention. At the very least, and without attempting to limit the application of the doctrine of equivalent ranges to the scope of the claims, each numerical parameter should at least be interpreted in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term "about" when used in conjunction with a stated value or range has the meaning that one skilled in the art reasonably ascribes to it, i.e., slightly more or less than the stated value or range is expressed as being within the following ranges: ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0518] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0519] Unless otherwise indicated herein or obviously contradictory to the context, the terms "one / kind", "said" and similar references used in the context of describing the present invention (especially in the context of the appended claims) should be interpreted as covering singular and plural references. The range of numerical values ​​listed herein is intended only as a convenient method for individually referring to each individual numerical value falling within the range. Unless otherwise indicated herein, each individual numerical value is incorporated into this specification as if each individual numerical value is individually listed herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or obviously contradictory to the context. Any and all embodiments or exemplary language (e.g., "such as") provided herein are only intended to better illustrate the present invention, without limiting the scope of the present invention claimed in other ways. The language in this specification should not be interpreted as indicating that any unclaimed element is necessary for practicing the present invention.

[0520] The grouping of alternative elements or embodiments of the present invention disclosed herein should not be interpreted as limiting. Each group member may be mentioned and claimed individually or in any combination with other members in the group or other elements found herein. For reasons of convenience and / or patentability, one or more members in the expected group may be included in the group or deleted from the group. When any such inclusion or deletion occurs, this specification is considered to include the modified group, therefore satisfying the written description of all Markush groups used in the appended claims.

[0521] Certain embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. Of course, variations of these described embodiments will be apparent to those of ordinary skill in the art after reading the foregoing description. The inventors expect that such variations will be adopted by those skilled in the art in due course, and the inventors expect that they can be implemented in a manner other than the manner specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter recited in the claims as permitted by the applicable law attached. In addition, unless otherwise indicated herein or clearly contradictory to the context, any combination of the above elements in all possible variations is encompassed within the present invention.

[0522] In addition, numerous references have been made throughout the specification to patents, printed publications, journal articles, and other written texts (materials cited herein). Each reference is individually incorporated herein by reference in its entirety for its teachings of reference.

[0523] Finally, it should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be used are also within the scope of the present invention. Thus, for example, but not limited to, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Thus, the present invention is not limited to the solutions explicitly shown and described.

[0524] The details shown herein are by way of example and for the purpose of illustrative discussion of the preferred embodiments of the invention only, and are presented in order to provide what is believed to be the most useful and understandable description of the principles and conceptual aspects of the various embodiments of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary for a basic understanding of the invention, and the description made with the aid of the drawings and / or examples makes it clear to those skilled in the art how several forms of the invention may be implemented in practice.

[0525] Unless clearly and unambiguously modified in the following examples or when the application of the meaning makes any construction meaningless or substantially meaningless, the definitions and explanations used in this disclosure are intended to control any future construction. In the case where the construction of a term would make it meaningless or substantially meaningless, the definition should be obtained from Webster's Dictionary, 3rd edition or dictionaries known to those of ordinary skill in the art such as the Oxford Dictionary of Biochemistry and Molecular Biology (Anthony Smith, ed., Oxford University Press, Oxford, 2004).

Claims

1. A method for genetically modifying a hematopoietic stem and progenitor cell (HSPC) population in a biological sample, comprising adding gold nanoparticles (AuNPs) to the biological sample, wherein the AuNPs comprise Gold (Au) core with a diameter less than 20nm; A clustered regularly interspaced short palindromic repeats (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 having a thiol modification, and the 5' end is conjugated to the nuclease, and wherein the thiol modification is covalently attached to the surface of the Au core, and wherein the crRNA has a sequence as shown 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 as shown in SEQ ID NO:48; SEQ ID NO:4; SEQ ID NO:15; SEQ ID NOs:33-41; SEQ ID NOs:44-47; or SEQ ID NOs:49-51; and CD133 targeting ligands comprising the binding domain of antibody clones REA820, REA753, REA816, 293C3, AC141, AC133 or 7 wherein the targeting ligand is linked to the nuclease via an amine-thiol crosslinker or a thiol-thiol crosslinker and wherein the HSPC population has not been exposed to electroporation, a viral vector encoding HDT, or a magnetic cell separation process, and wherein the method results in no more than 30% HSPC cytotoxicity and provides at least 10% gene editing efficiency within the HSPC population.

2. The method of claim 1, wherein the crRNA targets a sequence as shown 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 shown 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 for genetically modifying a selected cell population in a biological sample, comprising adding gold nanoparticles (AuNPs) to the biological sample, wherein the AuNPs comprise Gold (Au) core with a diameter less than 30nm; 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 having a chemical modification, and the 5' end is conjugated to the nuclease, and wherein the chemical modification is covalently attached 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; as well as A donor template comprising a homology directed repair template (HDT) on the surface of the positively charged polymer coating wherein the selected cell population is not exposed to electroporation or an encoded viral vector, and wherein the method results in no more than 30% cytotoxicity in the selected cell population and provides at least 10% gene editing efficiency within the selected cell population.

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.

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