Genetic modification protocols in immune cells

Abstract

The present invention relates to a method for genetic manipulation of immune cells or progenitors thereof, by lipid nanoparticles delivery of a gene modifying agent to cells.

Description

[0001] GENETIC MODIFICATION PROTOCOLS IN IMMUNE CELLS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of genetic manipulation of immune cells and progenitors thereof, wherein gene modifying agents are delivered to cells by means of lipid nanoparticles. In particular, the invention relates to the field of gene editing of immune cells and progenitors thereof.

[0004] BACKGROUND

[0005] Ex vivo gene editing of human T cells and hematopoietic stem / progenitor cells (HSPCs) holds therapeutic potential for the treatment of several diseases.

[0006] Among several editing strategies, nuclease-based approaches have been most investigated. They encompass transient delivery of nuclease mRNA or ribonucleoprotein (RNP) to introduce a DNA double strand break (DSB) at a target site. Cellular repair of the DSB can install genetic edits through non-homologous / microhomology-mediated end joining (NHEJ / MMEJ) or homology -driven recombination (HDR) using a DNA template often co-delivered by means of viral vectors, such as adeno-associated viral vectors (AAV) or integrase-defective lentiviral vectors (IDLV).

[0007] Whereas NHEJ allows disrupting gene coding frame or regulatory elements, HDR enables in situ correction of mutant alleles or targeted integration of a transgene cassette in safe harbors, such as AAVSl.

[0008] Further editing strategies include “prime editing”, a genome editing technique that mediates targeted insertions, deletions, or base-to-base conversions, using an engineered prime editing guide RNA (pegRNA) engaging a prime editor protein consisting of a Cas9 nickase fused to a reverse transcriptase, and “base editing”, wherein single-nucleotide chemical modifications are introduced in the genome of a cell through a guide RNA (gRNA) engaging a base editor protein that comprises a catalytically disabled nuclease or a Cas9 nickase fused to an enzyme capable of chemical modifying a DNA or RNA base.

[0009] Moreover, epigenetic editing strategies have been developed, wherein the epigenome of a cell is modified at specific sites using engineered epigenetic editors, comprising a DNA binding domain and an epigenetic effector domain, capable of modifying epigenomic features.

[0010] However, it has been shown that editing can activate robust and cumulative p53 -dependent DNA damage response (DDR), in particular upon sensing the nuclease-induced DSBs and / or the viral genome carrying the HDR template. This promotes cell cycle arrest and decreases hematopoietic reconstitution potential and graft clonality in xenotransplanted mice.

[0011] Moreover, gene editing agents are typically delivered to cells by electroporation, which induces physical permeabilization of cellular membranes and mitochondrial DNA damage, causing cytotoxicity that further contributes to the adverse impact of gene editing.

[0012] Efforts have been then focused on the provision of new means for delivering gene modifying agents, such as lipid nanoparticles (LNPs).

[0013] Geczy R et al. (Abstract 46 / Cytotherapy 24, 2022 and Abstract 1237 / Cytotherapy 25, 2023) disclose the exposure of expanded T cells to LNPs encapsulating Cas9 and sgRNAs for gene editing.

[0014] Furthermore, methods for improving gene-modification of cells by LNPs have been studied, including modifications of chemistry or size of LNPs, as well as new LNPs formulations, in order to avoid certain drawbacks of this delivery system, such as lower efficiency compared to electroporation.

[0015] WO 2020 / 206231 discloses a method for preparing genetically-modified immune cells, comprising contacting immune cells with LNPs comprising mRNA encoding an engineered nuclease, for modifying immune cells, together with an apolipoprotein, such as ApoE, which is said to improve the efficiency of the process and the properties of the resulting genetically- modified immune cells.

[0016] Still, the prior art struggles with problems of toxicity and / or low efficiency of genetic- modification protocols, that have not been overcome so far.

[0017] There is therefore the need for novel protocols for genetic modification of cells, in particular for gene editing of cells, having comparable efficiency as electroporation, but significantly lower toxicity.

[0018] Also, preservation of the long-term repopulating capacity of cells upon gene manipulation is highly desirable, especially for ex vivo gene editing in cells of the immune system, such as T cells, or of progenitors thereof, such as hematopoietic stem and progenitor cells (HSPCs), in the aim of using said gene edited cells as medicament.

[0019] BRIEF DESCRIPTION OF THE INVENTION

[0020] The limitations of the prior art are overcome by the present invention, providing a novel method for genetic modification of cells of the immune system, or progenitors thereof, delivering the gene-modifying agent by lipid nanoparticles (LNPs), said method comprising incubating the cell with LNPs comprising at least one gene-modifying agent, thus obtaining a genetically modified cell, and being characterized in that the culture medium wherein the cell is incubated with the LNPs is serum-free and in that LNPs are removed from the culture medium after incubation and prior to collection of the genetically modified cells.

[0021] Preferably, the genetic modification is gene editing and the gene-modifying agent delivered by the lipid nanoparticles is a gene editing agent, more preferably nuclease mRNAs or ribonucleoproteins (RNPs), capable of introducing a DNA double strand break (DSB) at a target site within the genome of the cell.

[0022] According to further preferred aspects, the genetic modification is prime editing or base editing, and the gene-modifying agent delivered by the lipid nanoparticles is a prime editor or a base editor, respectively.

[0023] According to further preferred aspects, the genetic modification is epigenetic editing, and the gene-modifying agent delivered by the lipid nanoparticles is an epigenetic editor.

[0024] The invention is also directed to a population of genetically modified cells, such as a population of edited cells, obtained by said method and to their use, as such or in a pharmaceutical formulation, as a medicament in a method of treatment of a disease.

[0025] The optimized conditions of the method of the invention provide lower toxicity compared to the methods of the prior art, with similar or even higher and prompter yields of genetically modified cells.

[0026] It is especially noted that removal of LNPs from the culture medium after incubation, according to the method of the invention, preserves cells growth over time and cell functionality, either in culture and in vivo.

[0027] Surprisingly, despite reducing the time of contact of LNPs with the cells, the method of the invention results to an overall increase of efficiency of genetic modification, compared to methods of the prior art wherein the LNPs are not removed. Clearly, this provides an efficient and cost-effective solution to the prior art limits.

[0028] Notably, the method of the invention also benefits ex vivo gene editing, better preserving the clonogenic potential of edited cells, in particular when cells are HSPCs.

[0029] Overall, the method of the invention then provides efficient and harmless delivery of genemodifying agents and, more generally, RNA-based therapeutics, in hematopoietic and immune cells.

[0030] Increase in cell recovery by reducing acute toxicity of genetically modified cells can improve the efficacy of immunotherapies by shortening manufacturing process and / or by manufacturing products otherwise not reaching the established therapeutic dose owing to harvest constraints. Moreover, minimizing the loss of genetically-modified T cells, allows to maintain a broader TCR repertoire diversity that may be crucial for some applications, such as immunotherapy. Similarly, reaching higher number of genetically-modified HSPCs can be pivotal to obtain highly polyclonal products and to provide safer therapeutic opportunities for patients yielding low / fragile HSPCs upon harvesting.

[0031] BRIEF DESCRIPTION OF FIGURES

[0032] Figure 1: A) Schematics of gene editing experimental procedure and multi-parametric analysis performed in CD4+ T cells. B) Percentage of HDR- and NHEJ-edited alleles after CD40LG editing with CRISPR / Cas9 RNP in absence (RNP) or presence of AAV6 (RNP+AAV6) in male CD4+ T cells (n=3). Median. UT: untreated cells; Mock Electro: mock electroporated cells; AAV6: AAV6 transduced-only cells, without electroporation. Mean ± SEM. CD4+ T cell phenotypes were defined as follows: Effector Memory RA (TEMRA): CD45RA+CD62L-; Effector Memory (EM): CD45RA-CD62L-; Central Memory (CM): CD45RA-CD62L+; T Memory Stem (TSCM): CD45RA+CD62L+. C) Percentage of (from lower to higher bar, in order) live, early apoptotic, late apoptotic and necrotic cells 24 h after editing (n = 3). Mean± SEM. D) Growth curve of CD4+ T cells (n=3). Median ± range.

[0033] Figure 2: A) Schematics of gene editing experimental procedure, and related analyses, after CRISPR / Cas9 RNA delivery by LNPs or CRISPR / Cas9 RNP / RNA delivery by electroporation in T cells. B) Percentage ofNHEJ-edited CD40LG alleles after editing male CD4+ T cells using 1.25 pg of LNPs, 25 pmol of RNP or 1.25 pg of RNA (n=6). Friedman test with Dunn’s multiple comparisons. Median ± interquartile range (IQR). C) Percentage of live, early / late apoptotic and necrotic cells 24 h after treatments in B and in UT cells (n = 6). Friedman test with Dunn’s multiple comparisons performed at live cells. Mean ± SEM. D) Relative Fluorescence Intensity (RFI) of Mitotracker Red (mitochondrial membrane potential) normalized on Mitotracker Green (mitochondrial mass) measured by flow cytometry 2 h (left) or 24 h (right) after indicated treatments (n=3,9,9). LNPs and electroporated conditions were pooled for this analysis. Wilcoxon’s test. Median ± IQR. E) Growth curve from C (n=6). Friedman test with Dunn’s multiple comparisons performed at day 3. Median ± IQR. F) Number of edited cells 24 h after editing in four experiments from C (n=4). Median ± IQR. G) Cell population composition from C (n=6), 14 days after treatment. Mean ± SEM. H) Percentage of live, early / late apoptotic and necrotic CD3+ T cells 24 h after B2M editing using 1.5 pg of LNPs, 50 pmol of RNP or 1.5 pg of RNA (n=3). UT cells were used as control. Mean ± SEM. I) Growth curve of CD3+ T cells from H (n=3). Median ± range. J) Percentage of B2M- cells (biallelic knockout) measured by flow cytometry within CD4+, CD8+ and total CD3+ T cell populations from H (n=3). Median ± range. K) Cell population composition from H (n=3), 14 days after treatment. Mean ± SEM. L) Number of B2M- cells from H 24 hours after editing (n=3). Median ± range. M) Percentage of reporter+ male CD4+ T cells after CD40LG editing using 1.25 pg of LNPs, 25 pmol of RNP or 1.25 pg of RNA, and the cognate HDR template provided by AAV6 (n=8,6,6). Kruskal- Wallis test followed by post hoc analysis with Dunn’s test. Median ± IQR N) Percentage of reporter+ cells within CD4+ T cells subpopulations from M (n=8,6,6). Kruskal-Wallis test followed by post hoc analysis with Dunn’s test. Median ± IQR. O) Percentage of live, early / late apoptotic and necrotic CD4+ T cells 24 h after editing in experiments from M. UT cells were used as controls (n=8,8,6,6). Kruskal-Wallis test followed by post hoc analysis with Dunn’s test on live cells. Mean ± SEM. P) Growth curve of CD4+ T cells from O. Kruskal-Wallis test followed by post hoc analysis with Dunn’s test performed at day 3. Median ± IQR. Q) Number of reporter+ cells from O, 24 hours after editing. Kruskal-Wallis test followed by post hoc analysis with Dunn’s test. Median ± IQR. R) Cell population composition from O (n=3), 14 days after treatment. Mean ± SEM. S) Percentage of live, early / late apoptotic and necrotic CD3+ T cells 24 h after AAVS1 editing using 1.25 pg of LNPs, 25 pmol of RNP or 1.25 pg RNA, and the cognate HDR template provided by AAV6 (n=3). UT cells were used as controls. Mean± SEM. T) Growth curve of CD3+ T cells from S (n=3). Median ± range. U) Percentage of GFP+ CD3+ T cells within CD4+, CD8+ and total populations from S (n=3). Median ± range. V) Number of GFP+ cells from R 24 hours after editing. Median ± IQR. W) Cell population composition from S (n=3), 14 days after treatment. Mean± SEM.

[0034] Figure 3: A) Percentage of T cells negative, single-, double-, triple, quadruple-positive for exhaustion markers 14 days after treatments (n=3). Mean± SEM. B) IFN-y secretion 3 days after anti-CD3 polyclonal stimulation, i.e., 14 days after treatments (n=3). Each symbol represents a different T cell donor.

[0035] Figure 4: A) Schematics of gene editing experimental procedure, and related analyses, after CRISPR / Cas9 RNA delivery by LNPs or CRISPR / Cas9 RNP / RNA delivery by electroporation in mPB-HSPCs. B) Percentage of B2M- HSPC within subpopulations (n=6). Cells were electroporated with 25 or 50 pmol of RNP, or transfected with 1, 1.5 or 2 pg of LNPs. mPB- HSPCs phenotype (from committed to primitive progenitors) is defined as follows: CD34-; CD34+CD133-; CD34+CD133+; CD34+CD133+CD90+. Friedman test with Dunn’s multiple comparisons performed on the latter subpopulation. Median ± IQR. C) Number of edited cells from B, 24 hours after editing (n=6). Friedman test with Dunn’s multiple comparisons. Median ± IQR. D) Percentage of live, early / late apoptotic and necrotic CD34+CD133+CD90+ HSPCs 24 h after B2M editing in experiments from B (n=6). Mock Electro and Empty LNPs were added as controls (n=6). Friedman test with Dunn’s multiple comparisons performed on live cells. Mean ± SEM. E) Growth curve of HSPCs from D (n=6). Median ± IQR. F) Cell population composition from D, 4 days after treatment. Mean ± SEM. G) Number of colonies generated by mPB HPSCs from D (n=6). Friedman test with Dunn’s multiple comparisons. Median ± IQR. H) Fold change expression of p21 (left) and APOBEC3H (right) relative to UT 24 hours after treatment from experiments in D (n=6). Friedman test with Dunn’s multiple comparisons. Median ± IQR. I) Percentage of circulating human (h)CD45+cells over time in mice transplanted with mPB-HSPCs edited with 50 pmol of RNP, 1.5pg of mRNA, or 1.5pg of LNPs (n=5,5,6). Kruskal -Wallis test. Median ± IQR. J) Percentage of hCD45+cells in BM and spleen of mice from I. Kruskal-Wallis test. Median. K) Percentage of B2M- cells over time, within human graft in PB of mice from I. Kruskal -Wallis test performed at the last time point. Median ± IQR. L) Percentage of B2M- cells within human graft in BM and spleen of mice from I. Kruskal -Wallis test. Median. M) Percentage of circulating human (h)CD45+cells over time in mice transplanted with CB-HSPCs edited with 50 pmol of RNP or 1.5 pg of LNPs (n=6,7). Mann-Whitney test. Median ± IQR. N) Percentage of hCD45+cells in BM and spleen of mice from M. Mann-Whitney test. Median. O) Percentage of B2M- cells over time, within human graft in PB of mice from N. Mann-Whitney test performed on the last time point. Median ± IQR. P) Percentage of B2M- cells within human graft in spleen of mice from M. Mann- Whitney test. Median. Q) Percentage (left) and MFI (right) of LDLR+ cells within mPB HSPC subpopulations 3 days after seeding (n=9). Friedman test with Dunn’s multiple comparisons. Median ± IQR. R) Percentage of B2M- cells within mPB HSPC subpopulations edited with different doses (pg) of RNA or LNP and then washed at different times (n = 1).

[0036] Figure 5: A) Schematics of gene editing experimental procedure, and related analyses, after CRISPR / Cas9 RNA delivery by LNPs or CRISPR / Cas9 RNP / RNA delivery by electroporation, in combination with the cognate AAV6, in mPB-HSPCs. B) Percentage of GFP+ cells within mPB-HSPC subpopulations 96 h after AAVS1 editing. Cells were edited with 25 pmol of RNP or 1 pg of LNPs and transduced with AAV6 immediately after electroporation or 2 h before transfection, respectively. Wilcoxon’s test on individual subpopulations. Median ± IQR. C) Percentage of live, early / late apoptotic and necrotic CD34+CD133+CD90+ mPB-HSPCs 24 h after AAVS1 editing in experiments from B (n=6). UT, Mock Electro and Empty LNPs were added as controls (n=6). Friedman test with Dunn’s multiple comparisons performed on live cells. Mean ± SEM. D) Growth curve from C (n=6). Median ± IQR. E) Number of edited cells from C, 24 h after editing (n=6). Wilcoxon’s test. Median ± IQR. F) Cell population composition from C (n=6), 4 days after treatment. Mean ± SEM. G) Number of colonies generated by mPB HPSCs from C (n=6). Median ± IQR. Friedman test with Dunn’s multiple comparisons. H) Fold change expression of p21 and APOBEC3H relative to UT 24 hours after treatment from experiments in C. Friedman test with Dunn’s multiple comparisons. Median ± IQR. I) Percentage of circulating hCD45+cells over time in mice transplanted with mPB- HSPCs (from a pool of donors) edited with 25 pmol RNP or 1 pg of LNPs, in combination with AAV6 (n=4,5). Median + range. J) Percentage of hCD45+cells in BM and spleen of mice from I. Median. K) Percentage of circulating hCD45+cells overtime in mice transplanted with mPB- HSPCs edited as in I, but using a different pool of donors (n=7). Mann-Whitney test. Median ± range. L) Percentage of hCD45+cells in BM and spleen of mice from K. Mann-Whitney test. Median. M-P) Percentage of GFP+ cells over time within human graft in PB (M, O) or hematopoietic organs (N, P) of mice from I (M, N) and K (O, P). Mann-Whitney test. Median or median + range.

[0037] Figure 6: A) Percentage of NHEJ- and HDR-edited CD40LG alleles after editing male CD4+ T cells with LNPs alone (NHEJ) or in combination with the cognate HDR template provided by AAV6 (HDR) in presence of different culture media. Good manufacturing practice culture protocol with Transact stimulation (GMP prot transact) (NHEJ: n=5, HDR: n=2); culture protocol with fetal bovine or human serum (FBS orHS) (NHEJ: n=4, HDR: n=2); GMP culture protocol with Dynabeads (GMP prot dynabeads) stimulation (HDR: n=2). Median ± IQR. B) Percentage (left graph) and MFI (right graph) of LDLR+ cells measured from the samples in A, by flow cytometry 3 days after seeding (n=l 5,6,2). Mann-Whitney test. Median ± IQR. C) Growth curve of male CD4+ T cells either UT or edited (GE) at CD40LG using different culture protocols (n=8,6,8,6). Median ± IQR. D) Percentage of NHEJ-edited CD40LG alleles after editing male CD4+ T cells with different doses of LNPs or RNA (n=2). One condition was washed 24 h after treatment. Electroporation of 25 pmol of RNP was used as control. Median. E) Percentage of live, early / late apoptotic and necrotic (from bottom to top of each bar) CD4+ T cells 24h after CD40LG editing in experiments from D (n=2). UT cells were used as controls. Mean. F) Percentage of viable cells over time (left) and growth curve (right) of CD4+ T cells from E (n=2). Median. G) Percentage of reporter+ CD4+ T cells after CD40LG editing using different doses LNPs or 1.25 pg of RNA electroporation in combination with AAV6 (n=2,2,2,l,2). Median. H-I) Percentage of reporter+ CD4+ T cells after CD40LG editing using 1.25 pg of LNPs in combination with AAV6 delivered at different time points before or after LNPs addition. 25 pmol of RNP and 1.25 pg of mRNA in combination with AAV6 delivered 15 min after electroporation were used as controls, as well as UT cells AAV6 only transduced conditions (left: n=l; right: n=3). Median ± IQR.

[0038] Figure 7: A) Percentage of circulating hCD45+ cells over time in mice transplanted with mPB- HSPCs edited with 25 pmol of RNP or 1.5pg of LNPs (n=7,6), targeting AAVS1. Mann- Whitney test. Median ± IQR. B) Percentage of hCD45+ cells in BM and spleen of mice from G (n=7,6). Mann-Whitney test. Median. C) Percentage of NHEJ-edited AAVS1 alleles within human cells in BM of mice from A. Mann-Whitney test. Median

[0039] Figure 8: Percentage of CD40L-edited T cells using IDLV as donor template (IDLV), with or without LentiBoost transduction enhancer (LB), with delivery of Cas9 RNA / RNP by electroporation (electro RNA, electro RNP) or by LNPs (LNPs).

[0040] Figure 9: A) Scheme of gene editing experimental procedure in mPB-derived HSPC. B) Percentage of HDR-edited cells after AAVSJ editing with CRISPR / Cas9 at different doses (low dose, LD = 0.5 pg; intermediate dose, ID = 1 pg; high dose, HD = 1.5 pg), in presence of AAV6, in HSPC cultured in research grade (StemSpan™ medium with research grade human cytokines; AAV 2xl04vg / cell) or clinically compliant (Cell growth medium with clinically compliant human cytokines; AAV 5xlO3vg / cell) conditions (n=l). C, D) Percentage of NHEJ-

[0041] (C) or HDR- (D) edited CD90+ HSPC incubated with commercial LNPs formulations optimized for HSCs (HSC1, HSC2, HSC3) and T cells (T cell) (n=4). Median with range.

[0042] Figure 10: A) Scheme of gene editing experimental procedure in mPB-derived HSPC in the presence of editing enhancers. B) Percentage of HDR-edited CD90+ HSPC after AAVS1 editing in presence (Enh.) or not (LNP +AAV) of editing enhancers. C, D) Fold change expression of p21 (C) and APOBEC3H (D) relative to untreated (UT) 24 hours after treatment, from experiments in A.

[0043] Figure 11: A) Scheme of base editing experimental procedure with ABE8.20-m RNA delivery by LNPs (LNP) or by electroporation (mRNA electro) in mPB-derived HSPC cells. B) Percentage of B2M negative cells within CD90+ cells from experiments in A. (n=4). Median with range.

[0044] Figure 12: A) Scheme of base editing experimental procedure in the context of HSC selection and expansion. B) Representative flow cytometry plot for the gating strategy used for the experiment in A. C-E) Percentage of B2M negative cells in the bulk (C), CD90 intermediate

[0045] (D) and CD90high (E) populations. (n=2). Median with range. F) Percentage of HSPC subpopulations after 7 days of expansion (n=l).

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0048] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of cells, such as a population of cells. The term “individual” or “subject” herein refers to a mammal, preferably human or non-human mammal, more preferably mouse, rat, other rodents, rabbit, dog, cat, pig, cow, horse or primate, most preferably human.

[0049] The terms “engineered” or “genetically modified”, as referred to cells, are herein used interchangeably.

[0050] “Engineering” or “genetic modification” or “genetic manipulation” of a cell according to the present invention include “gene transfer”, “gene editing” and “epigenetic editing”.

[0051] The term “gene transfer” refers to the transfer of genetic material (e.g., DNA or RNA) of interest into a cell, typically to treat or prevent a genetic or acquired disease or condition. For instance, the term “gene transfer” can refer to the addition of a copy of a gene into the genome of a cell, such as a correct copy of a gene that is completely or partially deleted or completely or partially not functional in the isolated cell (gene therapy). In particular, a genetic material of interest typically encodes a product (e.g., a protein polypeptide, peptide or functional RNA) whose production in vivo is desired. For example, a genetic material of interest can encode an enzyme, hormone, receptor, or polypeptide of therapeutic value. "Gene transfer" or "gene delivery" also refer to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells.

[0052] The term “gene editing” refers to the modification of the genome of a cell at a specific location to correct or alter a genetic sequence. The term “gene editing” refers in particular to a type of genetic engineering in which a nucleic acid is inserted, deleted or replaced in a cell. The term “gene editing” thus encompasses targeted disruption of a gene coding sequence, precise sequence substitution for in situ correction of mutations and targeted transgene insertion into a predetermined locus.

[0053] The term “epigenetic editing” refers to the modification of the epigenome of a cell at specific sites using engineered epigenetic editors composed of a DNA binding domain that target specific sequences in the epigenome and an effector domain that modifies epigenomic features. Epigenetic editing includes histone modification, as well as modification of DNA methylation or demethylation, or chromatin remodelling.

[0054] The term “gene-modifying agent”, as used herein, refers to an agent capable of modifying the genome or epigenome of a cell. The term “gene editing agent” or “gene editor” refers to an agent capable of editing a target site in the genome of a cell, e.g. by mediating disruption of the target site or correcting the target site.

[0055] The term “epigenetic editing agent” or “epigenetic editor” refers to an agent capable of editing a target site in the epigenome of a cell.

[0056] "Host cells," "cells", "cell lines," "cell cultures", “engineered cells” and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for gene modification include the original progeny of the original cell.

[0057] The terms “culture or culturing”, “growth or growing”, referred to cells, are used herein interchangeably and are meant to indicate maintenance of a cell population in vitro or ex vivo, preferably including expansion of the cell population.

[0058] The term “incubating” or “incubation”, as referred herein to cells and lipid nanoparticles according to the invention, means contacting a cell with a lipid nanoparticle and maintaining the contact for a time suitable for delivering the content of the lipid nanoparticles into the cell. The term “recombinant viral vector” or “viral vector” refers to an agent obtained from a virus through genetic engineering techniques capable of transferring genetic material (e.g., DNA or RNA) of interest to a cell, which results in production of the product encoded by that said genetic material (e.g., a protein polypeptide, peptide or functional RNA) in the target cell. Herein, the terms “transgene" refers to a transgene that is transferred to the recipient cell upon transduction of the virus. The term “viral vector” or “recombinant viral vector”, as used herein, also refers to the recombinant viral particles being a packaged viral vector, capable of binding to and entering recipient cells, delivering the vector transgene.

[0059] The term “engraftmenf ’ as used herein refers to the ability of cells to populate and survive in a subject following their transplantation, i.e., in the short and / or long term after transplantation. For example, engraftment may refer to the number and / or percentages of haematopoietic cells descended from transplanted haematopoietic stem and / or progenitor cells (e.g., graft-derived cells) that are detected about 1 day to 24 weeks, 1 day to 10 weeks, or 1-30 days or 10-30 days after transplantation.

[0060] The present invention is directed to a method for genetic modification of a cell of the immune system or a progenitor thereof, comprising the steps of:

[0061] - providing a cell of the immune system or a progenitor thereof,

[0062] - incubating in a culture medium said cell with lipid nanoparticles (LNPs) comprising at least one gene-modifying agent, obtaining a genetically modified cell, wherein said culture medium is serum-free;

[0063] - removing, after incubation, the LNPs;

[0064] - optionally collecting the genetically modified cell.

[0065] As used herein, the term “serum-free” refers to the liquid, solid, or liquid and solid culture medium, for the growth of cells in culture, that lacks or is substantially free from serum (e.g., fetal bovine serum, calf bovine serum).

[0066] Preferably, the serum-free culture medium does not comprise serum or comprises serum at a concentration (vol / vol) of less than about 0.5%, less than about 1%, less than about 1.25%, less than about 2.5%, less than about 5%, or less than about 10%. More preferably, the serum-free culture medium does not comprise any serum.

[0067] Preferably, the step of incubating the cell with lipid nanoparticles is carried out for an incubation time of from 8 to 48 hours, more preferably from 15 to 30 hours, most preferably about 24 hours. The LNPs are removed at the end of the incubation time.

[0068] Advantageously, in the method for genetic modification of the invention the incubation time can be reduced, so that the cells phenotype is better preserved (in particular the phenotype of stem cells), without detriment to the genetic modification efficiency.

[0069] Preferably, the LNPs are removed within 6 to 72 hours from the starting of incubation, more preferably within 12-48 hours from the starting of incubation.

[0070] In preferred embodiments, the cells are cultured in a serum-free medium for 12-48 hours before incubation with the lipid nanoparticles.

[0071] More preferably, the cells are cultured in a serum-free medium for 12-48 hours before incubation and for 12-24 hours during incubation with LNPs.

[0072] Preferably, cells are cultured in a serum-free medium at least from two days before incubation and at least for 24 hours during incubation with LNPs.

[0073] According to preferred aspects of the invention, the method for genetic modification of a cell of the immune system or a progenitor thereof, comprises the steps of:

[0074] - providing a cell of the immune system or a progenitor thereof,

[0075] - incubating in a serum-free culture medium said cell with lipid nanoparticles (LNPs) comprising at least one gene-modifying agent, preferably for 8-48 hours, more preferably for 15-30 hours, most preferably for about 48 hours, obtaining a genetically modified cell,

[0076] - removing, at the end of the incubation step, the LNPs;

[0077] - optionally collecting the genetically modified cell. The step of removing, after incubation, the LNPs can be carried out by washing the cells with a buffer, such as PBS or DPBS, or with fresh culture medium, preferably with serum-free fresh culture medium

[0078] Preferably, the gene-modifying agent that is delivered to a cell in the method of the invention is an RNA gene-modifying agent.

[0079] According to further preferred aspects, the gene-modifying agent that is delivered to a cell in the method of the invention is a DNA gene-modifying agent.

[0080] Preferably, the genetic modification according to the method of the invention is gene editing or epigenetic editing and the gene-modifying agent is a gene editing agent or an epigenetic editing agent, respectively.

[0081] According to preferred aspects of the invention, the genetic modification is gene editing, more preferably non-homologous end joining (NHEJ) gene disruption, homology-driven repair (HDR) gene correction, prime editing or base editing. More preferably, the genetic modification is gene editing, more preferably non-homologous end joining (NHEJ) gene disruption, homology -driven repair (HDR) gene correction. According to further preferred aspects of the invention, the genetic modification is epigenetic editing.

[0082] Preferably, the gene-modifying agent according to the method of the invention is a gene editing agent, more preferably comprising or consisting of an engineered nuclease, or a polynucleotide encoding therefor. Preferred nucleases suitable for genome editing according to the present invention include zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease (Gaj, T. et al. (2013) Trends Biotechnol. 31: 397-405). Meganucleases (Silve, G. et al. (2011) Cur. Gene Then 11: 11-27) may also be employed as suitable nucleases for gene editing.

[0083] According to particularly preferred aspects of the invention, the gene-modifying agent delivered to a cell in the method of the invention is a gene editing agent being a transcribed mRNAs encoding a nuclease (nuclease mRNA) or a ribonucleoprotein (RNP) comprising a nuclease, more preferably said nuclease being selected from; zinc-finger nuclease, a transcription activator like effector nuclease (TALENs), and a CRISPR / Cas nuclease.

[0084] Therefore, according to preferred aspects of the invention, the lipid nanoparticles comprise a nuclease mRNA or a ribonucleoprotein (RNP) comprising a nuclease, having specificity for a recognition sequence in the genome of the immune cells. Contacting the cells with the lipid nanoparticles results in the delivery of the engineered nuclease encoding mRNA into the immune cells where the engineered nuclease is expressed. “CRISPR / Cas system”, or shortly “CRISPR system” or “CRISPR complex”, refer collectively to transcripts and other elements involved in the expression of, or directing the activity of, CRISPR-associated (“Cas”) nuclease, including DNA encoding a Cas nuclease, or an RNA transcript thereof, and a guide RNA, wherein the guide RNA (gRNA or sgRNA) may be selected to enable a Cas to be targeted to a specific sequence in the genome of a cell (van der Oost et al. (2014) Nat. Rev. Microbiol. 12: 479-92). Typically, in the context of formation of a CRISPR complex, “target sequence” generally refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and a guide sequence promotes the formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, the target sequence may be within an organelle of the cell.

[0085] According to preferred aspects of the invention, the gene editing agent is a CRISPR system comprising a non-coding RNA molecule (guide RNA, or gRNA), which sequence-specifically binds to DNA, and a Cas protein (e.g., Cas9), with nuclease functionality (e.g., two nuclease domains), that are comprised in the same of different LNPs.

[0086] Therefore, according to preferred aspects, the invention is directed to a method for genetic modification of a cell of the immune system or a progenitor thereof, comprising the steps of:

[0087] - incubating in a culture medium said cell with lipid nanoparticles (LNPs) comprising a Cas nuclease RNA and gRNA, more preferably comprising a Cas9 / gRNA ribonucleoprotein (RNP) complex, obtaining a genetically modified cell, wherein said culture medium is serum-free;

[0088] - removing, after incubation, the LNPs;

[0089] - optionally collecting the genetically modified cell.

[0090] Methods for the design of gRNAs are known in the art. Furthermore, fully orthogonal Cas9 proteins, as well as Cas9 / gRNA ribonucleoprotein complexes and modifications of the gRNA structure / composition to bind different proteins, have been recently developed to simultaneously and directionally target different effector domains to desired genomic sites of the cells (Esvelt et al. (2013) Nat. Methods 10: 1116-21), and are suitable for use in the invention.

[0091] Generally, an exogenous polynucleotide template may be used for recombination into the targeted locus comprising the target sequences. This is referred to as an “editing template” (or “donor template” or “editing (template) polynucleotide” or “editing sequence”).

[0092] In some aspects, the recombination is homologous recombination. The editing template can be delivered to a cell by means of a viral vector or by means of further lipid nanoparticles.

[0093] In preferred aspects of the invention, where the genetic modification according to the method of the invention is gene editing and the gene-modifying agent delivered by LNPs is a gene editing agent, the method further comprises a step of transducing a viral vector in a cell, more preferably an adeno-associated vector (AAV) or a retroviral vector, most preferably a lentiviral vector, or an integration-defective lentiviral vector (IDLV), delivering an editing template. Therefore, the method of the invention can further comprise a step of transducing the cells with a viral vector, preferably an adeno-associated vector (AAV), a lentiviral vector, or an integration-defective lentiviral vector (IDLV), preferably said viral vector comprising an editing template.

[0094] In some such embodiments of the method, the editing template is introduced into the cells during, before, or after, incubation with the lipid nanoparticles.

[0095] Optionally, the method further comprises a step of transducing of a viral vector in a cell, more preferably an adeno-associated vector (AAV) or a retroviral vector, most preferably a lentiviral vector, or an integration-defective lentiviral vector (IDLV), comprising a polynucleotide encoding a transgene, such as a transgene for gene therapy.

[0096] In preferred embodiments, the method of the invention further comprises a step of transducing the cell with a viral vector, preferably comprising an editing template, wherein the cell is contacted with the viral vector at the same time as it is contacted with the lipid nanoparticles, or within a time of from 1 minute up to 48 hours after the cell is contacted with the lipid nanoparticles, more preferably 12-48 hours after the cells are contacted with the lipid nanoparticles. In some embodiments, the cells are transduced with the viral vector within 12 hours, within 8 hours, or within 2 hours, prior to incubation of said cells with said lipid nanoparticles.

[0097] In further embodiments, the method of the invention further comprises a step of incubating the cell with lipid nanoparticles comprising an editing template.

[0098] Further preferred gene-modifying agents, according to the method of the invention, include prime editors, or base editors, or polynucleotides encoding therefor.

[0099] Non limiting examples of prime and base editors are / those described in: Gaudelli NM et al. Nat Biotechnol. 2020 Jul;38(7):892-900; Zeng J, et al. Nat Med. 2020 Apr;26(4):535-54I, Everette, K.A et al. Nat. Biomed. Eng 7, 616-628 (2023). According to further preferred aspects of the invention, the gene-modifying agent is an epigenetic agent, more preferably an artificial transcription repressor (ATR), or a polynucleotide encoding therefor.

[0100] Preferred ATRs suitable for epigenetic editing according to the present invention, include those described in WO2016063264A1, such as: an ATR comprising aDNA-binding domain operably linked to a KRAB domain; an ATR comprising a DNA-binding domain operably linked to a DNMT3A, DNMT3B or DNMT1 domain; an ATR comprising a DNA-binding domain operably linked to a DNMT3L domain; an ATR comprising a DNA-binding domain operably linked to a SETDB1 domain; and mixtures thereof; or polynucleotides encoding therefor.

[0101] Optionally, the method of the invention comprises incubating the cells with different LNPs, comprising different gene-modifying agents, such as different editing reagents and nucleic acids, wherein the different LNPs are present concurrently or asynchronously.

[0102] Multi-step delivery may allow multiplex editing while minimizing the risk of translocations. Preferably, when the method of the invention comprises a step of transducing the isolated cells with a viral vector, the transduction step includes stimulation of cells in the presence of a human cytokine mix (preferably IL-6, TPO, SCF, and FLT3-1), more preferably for about 22 -hour, followed by the addition of the viral particles, preferably for about 14 hours upon transduction. Preferably, when the method of the invention comprises a step of transducing the isolated cells with a viral vector, at least one viral transduction enhancer, more preferably PGE, CsH, CsA or a pol oxamer, and / or at least one expansion enhancer, more preferably UM171, UM729, StemRegeninl (SRI), diethylaminobenzaldehyde (DEAB), LG1506, BIO (GSK30 inhibitor), NR- 101, trichostatin A (TSA), garcinol (GAR), valproic acid (VP A), copper chelator, tetraethylenepentamine, and nicotinamide, is added to the cell culture before transduction, according to optimized protocols, for instance as described in WO2013049615, WO2018193118, WO2013127964 and in Delville et al.

[0103] The method of the invention is preferably carried out in vitro or ex vivo.

[0104] According to preferred embodiments of the invention, the cell to be genetically modified in the method of the invention, is a T cell or a hematopoietic stem and / or progenitor cell (HSPC). Preferably the cell is a mammalian cell, more preferably, the cells is a human cell.

[0105] The cell can be a mobilised peripheral blood cell, a cord blood cell, or a bone marrow cell. According to preferred aspects of the invention, the cells that undergo genetic modification in the method of the present invention are HSPCs. More preferably, said cells are CD34+ cells, and / or CD34+CD133+ cells, most preferably CD34+CD133+CD90+ cells.

[0106] According to further preferred aspects of the invention the cells that undergo genetic modification in the method of the present invention are T cells, more preferably CD3+ and / or CD4+ T cells.

[0107] Preferably, the cell is provided after selection and / or expansion of a population of cells; more preferably the cell is provided after selection and / or expansion of a specific population of cells, such as a population of CD4+ T cells or CD34+ HSPCs.

[0108] Therefore, preferably, the method of the invention comprises a step of selecting and expanding ex vivo a population of cells isolated from a subject, prior to incubating said cells with LNPs and / or to transducing said cells with a virial vector.

[0109] More preferably, the method of the invention comprises as a step of selecting and expanding ex vivo CD34+ HSPCs obtained and isolated by leukapheresis or bone marrow harvest, or a step of selecting and expanding ex vivo CD4+ T cells obtained and isolated from peripheral blood, prior to incubating said cells with LNPs and / or transducing the cells with a virial vector.

[0110] The method of the invention can be carried out in small (e.g., l-2xl06cells with lipid nanoparticles, at 2 days after selection) medium (e.g., 5-10xl06cells with lipid nanoparticles at 2 days after selection), or large scale (e.g., 100-500xl06cells with lipid nanoparticles at 2 days after selection).

[0111] For carrying out a small or medium scale of the method of the invention, for genetically modifying a CD4+ T cell according to preferred embodiments, CD4+ T-cells can be isolated, e.g., with microbeads, from buffy coats of healthy donors or from patients’ peripheral blood samples.

[0112] For carrying out a large scale of the method of the invention, for genetically modifying a CD4+ T cell according to preferred embodiments of the invention, CD4+ T-cells can be isolated from healthy donors’ (HD) leukaphereses.

[0113] The isolated cells are preferably activated and expanded for about 2 days before the step of genetic modification by incubating the cells with LNPs, more preferably before viral vector transduction of a donor template prior to incubating the cells with LNPs.

[0114] In certain embodiments of the method of the invention, the at least one gene modifying agent targets a gene selected from the group consisting of: CD40LG (CD40 Ligand), B2M, AAVSI, RAG1, UBA1, CXCR4, IL2RG, CYBB, HBB, WAS, RPS19, FANCA, DKC1, TRAC, TRBC, PD1, TIM3, and LAG3 genes. Preferably, the gene is a mammalian gene, more preferably a human gene.

[0115] Preferably, the invention is directed also to an ex vivo or in vitro method for genetic modification of a CD4+ T cell, comprising the steps of:

[0116] - providing a CD4+ T cell, more preferably after selecting and expanding the CD4+ T cell; - transducing said cell with an IDLV comprising a donor template, more preferably encoding for a splice acceptor sequence and the cDNA of CD40LG exons 2-5,

[0117] - incubating, in a serum-free culture medium, said cell with lipid nanoparticles (LNPs) comprising a genetic modifying agent, more preferably comprising a Cas9 nuclease, or a polynucleotide encoding therefor, and a gRNA targeting the first intron of CD40LG gene, obtaining a genetically modified cell;

[0118] - removing, after incubation, the LNPs;

[0119] - optionally collecting the genetically modified cell.

[0120] According to further preferred embodiments, the invention is directed to a GMP-compliant ex vivo method for genetic modification of a CD4+ T cell, comprising the steps of:

[0121] - at day 0, selecting CD4+ cells isolated from buffy coats of healthy donors;

[0122] - at day 6, enriching LNGFR+ cells, preferably with an automated cell manufacturing platform in a closed system (such as CliniMACS Prodigy ®), wherein cells are kept in a closed system with the sole exception of cell wash and centrifugation steps;

[0123] - optionally electroporating cells using a GMP-compliant assembly (such as R-1000 and R-20K process assemblies for the MaxCyte GTx electroporator), using 100-500xl06cells / run;

[0124] - incubating, in a serum-free culture medium, said cells with lipid nanoparticles (LNPs) comprising a genetic modifying agent, obtaining a genetically modified cell;

[0125] - removing, after incubation, the LNPs;

[0126] - optionally collecting the genetically modified cell.

[0127] Such method allows to obtain genetically modified CD4+cells whose purity is maintained until the end of the process with no expansion of CD8+ T cells.

[0128] According to preferred embodiments of the method of the invention, the cells are further genetically engineered to express an engraftment enhancer. More preferably said engraftment enhancer is CD47 and / or C-X-C chemokine receptor type 4 (CXCR4).

[0129] Then, according to preferred aspects of the invention, the method further comprises transfecting or transducing the cells with one or more vector encoding the CD47 and / or CXCR4; the CD47 and CXCR4 may be, for example, encoded on separate vectors or on the same vector. The vector can be a plasmid or a viral vector. Preferably, CD47 and / or CXCR4 are overexpressed in the cell. Optionally, RNA encoding the CD47 and / or CXCR4, or CD47 and / or CXCR4 protein, is introduced into the cells.

[0130] According to preferred embodiments, the method of the invention also comprises culturing the cells in the presence of an inhibitor of senescence, such as inhibitor of MAPK / ERK signaling, an IL-1 inhibitor and / or an NF-KB inhibitor. Preferably, the inhibitor of MAPK / ERK signaling is a MAP3K inhibitor, a MAK2K inhibitor, a MAPK inhibitor, preferably an MKK7 inhibitor, an MKK4 inhibitor, an MKK3 / 6 inhibitor, an MEK1 / 2 inhibitor, a JNK inhibitor, a p38 inhibitor, a p53 inhibitor or an ERK inhibitor. Preferably, the inhibitor of p53 activation is a p53 dominant negative peptide, an ataxia telangiectasia mutated (ATM) kinase inhibitor or an ataxia telangiectasia and Rad3 -related protein (ATR) inhibitor. In some embodiments, the inhibitor of p53 activation is pifithrin-a or a derivative thereof, KU-55933 or a derivative thereof; GSE56 or a variant thereof; KU-60019, BEZ235, wortmannin, CP-466722, Torin 2, CGK 733, KU-559403, AZD6738 or derivatives thereof; or an siRNA, shRNA, miRNA or antisense DNA / RNA, preferably wherein the inhibitor of p53 activation is GSE56 or a variant thereof.

[0131] LNPs that are used in the method of the invention can comprise cationic and / or non-cationic lipids.

[0132] Preferably, the LNPs used in the method of the invention comprise cationic lipids, more preferably selected from: DLin-DMA (l,2-dilinoleyloxy-3 -dimethylaminopropane), DLin- MC3-DMA (dilinoleylmethyl-4-dimethylaminobutyrate), DLin-KC2-DMA (2,2-dilinoleyl-4- (2- dimethylaminoethyl)-[l,3]-dioxolane), DODMA (1,2- dioleyloxy-N,N-dimethyl-3- aminopropane), SS-OP (Bis[2-(4-{2-[4-(cis-9- octadecenoyloxy)phenylacetoxy]ethyl}piperidinyl)ethyl] disulfide), and derivatives thereof. Additional and non-limiting examples of cationic lipids include methylpyridiyl- dialkyl acid (MPDACA), palmitoyl-oleoyl-nor-arginine (PONA), guanidino-dialkyl acid (GUADACA), l,2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2- dioleoyl-3- trimethylammonium-propane (DOTAP), Bis{2-[N-methyl-N-(a-D- tocopherolhemisuccinatepropyl)amino]ethyl} disulfide (SS-33 / 3AP05), Bis{2-[4-(a-D- tocopherolhemisuccinateethyl)piperidyl] ethyl} disulfide (SS33 / 4PE15), Bis{2-[4-(cis-9- octadecenoateethyl)-l-piperidinyl] ethyl} disulfide (SS18 / 4PE16), Bis{2-[4-(cis,cis-9,12- octadecadienoateethyl)-l-piperidinyl] ethyl} disulfide (SS18 / 4PE13), and mixtures thereof.

[0133] In some embodiments, the molar concentration of the cationic lipid is from 20% to 80%, from 30% to 70%, from 40% to 60%, from 45% to 55%, of the total lipid molar concentration.

[0134] According to preferred embodiments of the invention, the lipid nanoparticles comprise cationic lipids and an mRNA gene-modifying agent, in a molar ratio of cationic lipid to mRNA of from 1 to 20, from 2 to 16, from 4 to 12, from 6 to 10. In certain embodiments of the method, the lipid nanoparticles comprise a molar ratio of cationic lipid to mRNA of about 8.

[0135] Preferably, cells are incubated with LNPs in the presence of an apolipoprotein. Preferably, the apolipoprotein is an apolipoprotein A (ApoA), apolipoprotein B (ApoB), apolipoprotein C (ApoC), apolipoprotein D (ApoD), apolipoprotein E (ApoE), apolipoprotein H (ApoH), apolipoprotein L (ApoL), apolipoprotein M (ApoM), or apolipoprotein (a) (Apo(a)) protein. More preferably, the apolipoprotein is ApoE, such as ApoE2, ApoE3, or ApoE4.

[0136] Preferably, the LNPs utilized in the method of the invention comprise non-cationic lipids. Non-cationic lipids include, in some embodiments, phospholipids and steroids.

[0137] Phospholipids useful for the lipid nanoparticles described herein include, but are not limited to,

[0138] 1.2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-Didecanoyl-sn-glycero-3- phosphocholine (DDPC), l,2-Dierucoyl-sn-glycero-3-phosphate(Sodium Salt) (DEPA-NA),

[0139] 1.2-Dierucoyl-sn-glycero-3 -phosphocholine (DEPC), l,2-Dierucoyl-sn-glycero-3- phosphoethanolamine (DEPE), l,2-Dierucoyl-sn-glycero-3 [Phospho-rac-(l-glycerol)(Sodium Salt) (DEPG-NA), l,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), 1,2-Dilauroyl-sn- glycero-3-phosphate(Sodium Salt) (DLPA-NA), l,2-Dilauroyl-sn-glycero-3 -phosphocholine (DLPC), l,2-Dilauroyl-sn-glycero-3 -phosphoethanolamine (DLPE), 1,2-Dilauroyl-sn- glycero- 3[Phospho-rac-(l-glycerol...)(Sodium Salt) (DLPG-NA), 1,2-Dilauroyl-sn-glycero- 3[Phospho-rac-(l-glycerol)(Ammonium Salt) (DLPG-NH4), l,2-Dilauroyl-sn-glycero-3- phosphoserine(Sodium Salt) (DLPS-NA), l,2-Dimyristoyl-sn-glycero-3-phosphate(Sodium Salt) (DMPA-NA), l,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-Dimyristoyl- sn-glycero-3-phosphoethanolamine (DMPE), l,2-Dimyristoyl-sn-glycero-3[Phospho-rac-(l- glycerol)(Sodium Salt) (DMPG-NA), l,2-Dimyristoyl-sn-glycero-3[Phospho-rac-(l- glycerol)( Ammonium Salt) (DMPG-NH4), l,2-Dimyristoyl-sn-glycero-3[Phospho-rac-(l- glycerol)(Sodium / Ammonium Salt) (DMPG-NH4 / NA), l,2-Dimyristoyl-sn-glycero-3- phosphoserine(Sodium Salt) (DMPS-NA), l,2-Dioleoyl-sn-glycero-3-phosphate(Sodium Salt) (DOPA-NA), l,2-Dioleoyl-sn-glycero-3 -phosphocholine (DOPC), 1,2-Dioleoyl-sn- glycero-3- phosphoethanolamine (DOPE), l,2-Dioleoyl-sn-glycero-3[Phospho-rac-(l- glycerol)(Sodium Salt) (DOPG-NA), l,2-Dioleoyl-sn-glycero-3-phosphoserine(Sodium Salt) (DOPS-NA), 1,2- Dipalmitoyl-sn-glycero-3-phosphate(Sodium Salt) (DPPA-NA), 1,2- Dipalmitoyl-sn-glycero- 3 -phosphocholine (DPPC), 1 ,2-Dipalmitoyl-sn-glycero-3 -phosphoethanolamine (DPPE), 1 ,2- Dipalmitoyl-sn-glycero- 3[Phospho-rac-(l-glycerol)(Sodium Salt) (DPPG-NA), 1,2- Dipalmitoyl-sn-glycero- 3[Phospho-rac-(l-glycerol)(Ammonium Salt) (DPPG-NH4), 1,2- Dipalmitoyl-sn-glycero-3- phosphoserine(Sodium Salt) (DPPS-NA), 1,2-Distearoyl-sn- glycero-3-phosphate(Sodium Salt) (DSPA-NA), l,2-Distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 1,2- Distearoyl-sn-glycero-3[Phospho-rac-(l-glycerol)(Sodium Salt) (DSPG-NA), 1,2-Distearoyl- sn-glycero-3[Phospho-rac-(l-glycerol)(Ammonium Salt) (DSPG-NH4), 1,2-Distearoyl-sn- glycero-3-phosphoserine(Sodium Salt) (DSPS-NA), Egg-PC (EPC), Hydrogenated Egg PC (HEPC), Hydrogenated Soy PC (HSPC), 1-Myristoyl-sn-glycero- 3 -phosphocholine (LY S OPCM YRIS TIC ) , l-Palmitoyl-sn-glycero-3-phosphocholine (LYSOPCPALMITIC), 1- Stearoyl-sn-glycero-3-phosphocholine (LYSOPC STEARIC), 1- Myristoyl-2-palmitoyl-sn- glycero3 -phosphocholine (MPPC), l-Myristoyl-2-stearoyl-sn- glycero-3 -phosphocholine (MSPC), l-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), l-Palmitoyl-2- oleoyl-sn-glycero-3 -phosphocholine (POPC), l-Palmitoyl-2-oleoyl-sn- glycero-3- phosphoethanolamine (POPE), l-Palmitoyl-2-oleoyl-sn-glycero-3[Phospho-rac-(l- glycerol)] (Sodium Salt) (POPG-NA), l-Palmitoyl-2-stearoyl-sn-glycero-3 -phosphocholine (PS PC), l-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), l-Stearoyl-2-oleoyl- sn-glycero-3 -phosphocholine (SOPC), l-Stearoyl-2-palmitoyl-sn-glycero-3- phosphocholine (SPPC), and mixtures thereof. In particular embodiments, the phospholipid is DSPC. In particular embodiments, the phospholipid is DOPE. In particular embodiments, the phospholipid is DOPC.

[0140] Steroids useful for the lipid nanoparticles described herein include, but are not limited to, cholestanes such as cholesterol, cholanes such as cholic acid, pregnanes such as progesterone, androstanes such as testosterone, and estranes such as estradiol. Further steroids include, but are not limited to, cholesterol (ovine), cholesterol sulfate, desmosterol-d6, cholesterol-d7, lathosterol-d7, desmosterol, stigmasterol, lanosterol, dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, zymosterol-d5, 14-demethyl-lanosterol, 14-demethyl-lanosterol-d6, 8(9)- dehydrocholesterol, 8(14)-dehydrocholesterol, diosgenin, DHEA sulfate, DHEA, lanosterol- d6, dihydrolanosterol-d7, campesterol-d6, sitosterol, lanosterol-95, Dihydro FF- MAS-d6, zymostenol-d7, zymostenol, sitostanol, campestanol, campesterol, 7- dehydrodesmosterol, pregnenolone, sitosterol-d7, Dihydro T-MAS, Delta 5-avenasterol, Brassicasterol, Dihydro FF-MAS, 24-methylene cholesterol, cholic acid derivatives, cholesteryl esters, glycosylated sterols, and mixtures thereof. In preferred embodiments, the lipid nanoparticles comprise cholesterol.

[0141] In some embodiments of the method, the molar concentration of the non-cationic lipids is from 20% to 80%, from 30% to 70%, from 40% to 60%, from 45% to 55%, of the total lipid molar concentration.

[0142] In some embodiments of the method, the non-cationic lipids comprise a phospholipid, preferably DSPC, wherein the molar concentration of the phospholipid is from 0.1% to 30%, from 2.5% to 25%, from 5% to 20%, from 5% to 15%, from 7.5% to 12.5%, from 10% to 20% of the total lipid molar concentration.

[0143] In some embodiments of the method, the non-cationic lipids comprise a steroid, preferably cholesterol, wherein the molar concentration of the steroid is from 20% to 60%, from 25% to 55%, from 30% to 50%, from 35% to 40%, or 38.5% of the total lipid molar concentration.

[0144] In some embodiments of the method, the lipid nanoparticles comprise a lipid conjugate.

[0145] In some embodiments of the method, the lipid nanoparticles comprise non-cationic lipids and a lipid conjugate.

[0146] Such lipid conjugates include, but are not limited to, ceramide PEG derivatives such as C8 PEG2000 ceramide, C16 PEG2000 ceramide, C8 PEG5000 ceramide, C16 PEG5000 ceramide, C8 PEG750 ceramide, and C 16 PEG750 ceramide, phosphoethanolamine PEG derivatives such as 16:0 PEG5000 PE, 14:0 PEG5000 PE, 18:0 PEG5000 PE, 18: 1 PEG5000 PE, 16:0 PEG3000 PE, 14:0 PEG3000 PE, 18:0 PEG3000 PE, 18: 1 PEG3000 PE, 16:0 PEG2000 PE, 14:0 PEG2000 PE, 18:0 PEG2000 PE, 18: 1 PEG2000 PE 16:0 PEG1000 PE, 14:0 PEG1000 PE, 18:0 PEG1000 PE, 18:1 PEG 1000 PE, 16:0 PEG750 PE, 14:0 PEG750 PE, 18:0 PEG750 PE, 18:1 PEG750 PE, 16:0 PEG550 PE, 14:0 PEG550 PE, 18:0 PEG550 PE, 18:1 PEG550 PE, 16:0 PEG350 PE, 14:0 PEG350 PE, 18:0 PEG350 PE, and 18:1 PEG350, sterol PEG derivatives such as Chol-PEG600, and glycerol PEG derivatives such as DMG-PEG5000, DSG-PEG5000, DPG- PEG5000, DMG-PEG3000, DSG-PEG3000, DPG-PEG3000, DMG-PEG2000, DSG- PEG2000, DPG-PEG2000, DMG-PEG1000, DSG-PEG1000, DPG-PEG1000, DMG- PEG750, DSG-PEG750, DPG-PEG750, DMG-PEG550, DSG-PEG550, DPG-PEG550, DMG- PEG350, DSG-PEG350, DPG-PEG350, and mixtures thereof. In some embodiments, the lipid conjugate is a DMG-PEG.

[0147] Preferably, the lipid nanoparticles comprise a lipid conjugate being a pegylated lipid, more preferably a DMG-PEG, such as DMG-PEG2000 or DMG-PEG5000.

[0148] In some embodiments of the method, the molar concentration of the lipid conjugate is from 0.01% to 10%, from 0.2% to 8%, from 0.5% to 5%, from 0.1% to 1.5%, from 1% to 2%, of the total lipid molar concentration.

[0149] As used herein, the total lipid molar concentration is the sum of the cationic lipid, the noncationic lipid, and the lipid conjugate molar concentrations.

[0150] In some embodiments, a molar ratio of the cationic lipid to the phospholipid is from 1 : 1 to 20: 1, 2:1 to 20:1, 3:1 to 20: 1, 4: 1 to 20:1, 6:1 to 20:1, 8:1 to 20: 1, 10:1 to 20:1, 12: 1 to 20: 1, 14:1 to 20: 1, 16:1 to 20:1, 18: 1 to 20:1, or 2: 1 to 7: 1. In some embodiments, a molar ratio of the cationic lipid to the steroid is from 0.25:1 to 5:1, 0.5:1 to 5:1, 0.75: 1 to 5: 1, l: l to 5:1, 2:1 to 5: 1, 3: 1 to 5: 1, 4:1 to 5:1, 0.5:1 to 3: 1, or 0.8:1 to 2:1. In some embodiments, the molar ratio of the cationic lipid to the steroid is from 0.8: 1 to 2:1. In some embodiments, the molar ratio of the cationic lipid to the steroid is 0.8:1, 1.3:1, 1: 1, or 2:1. In some embodiments, a molar ratio of the cationic lipid to the lipid conjugate is from 10: 1 to 1000: 1, 25: 1 to 1000:1, 50:1 to 1000:1, 75: 1 to 1000:1, 100:1 to 1000:1, 250:1 to 1000: 1, 400: 1 to 1000: 1, 550: 1 to 1000: 1, 700: 1 to 1000: 1, 850: 1 to 1000: 1, 20:1 to 600:1, or 25:1 to 400:1. In some embodiments, the molar ratio of the cationic lipid to the lipid conjugate is from 25: 1 to 400:1.

[0151] In some embodiments, a molar ratio of the steroid to the lipid conjugate is from 5: 1 to 750: 1, 25:1 to 750: 1, 50: 1 to 750:1, 75:1 to 750:1, 100:1 to 750: 1, 150: 1 to 750:1, 200:1 to 750: 1, 250: 1 to 750:1, 300: 1 to 750: 1, 350:1 to 750:1, 400: 1 to 750:1, 450:1 to 750:1, 500:1 to 750: 1, 550: 1 to 750: 1, 600:1 to 750:1, 650:1 to 750:1, 700:1 to 750:1, 10: 1 to 500: 1, or 25:1 to 500: 1. In some embodiments, the molar ratio of the steroid to the lipid conjugate is from 25: 1 to 500: 1. In some embodiments, the molar ratio of the steroid to the lipid conjugate is from 25:1, 30: 1, or 500:1.

[0152] In some embodiments, a molar ratio of the phospholipid to the lipid conjugate is from 1 : 1 to 300: 1, 5:1 to 300:1, 10:1 to 300:1, 15:1 to 300:1, 20:1 to 300: 1,25: 1 to 300:1, 50: 1 to 300: 1, 75:1 to 300:1, 100:1 to 300:1, 125: 1 to 300:1, 150: 1 to 300: 1, 175: 1 to 300: 1, 200: 1 to 300: 1, 225: 1 to 300: 1, 250:1 to 300:1, 275: 1 to 300: 1, 3: 1 to 200: 1, or 5:1 to 100:1. In some embodiments, the molar ratio of the phospholipid to the lipid conjugate is from 5:1 to 100:1. In some embodiments, the molar ratio of the phospholipid to the lipid conjugate is 6: 1 , 10: 1, 13:1 or 100:1.

[0153] The selection of cationic lipids, non-cationic lipids and / or lipid conjugates comprised in the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, and the characteristics of the genetic-modifying agent to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios of each individual component may be adjusted accordingly.

[0154] The lipid nanoparticles for use in the method of the invention can be prepared by various techniques which are presently known in the art.

[0155] Selection of the appropriate size of lipid nanoparticles must take into consideration the site of the target cell and the application for which the lipid nanoparticles is being made.

[0156] Preferably, the lipid nanoparticles of the method of the invention have a median size of 25-500 nm, more preferably of 50-300 nm, most preferably of 80-150 nm.

[0157] A variety of methods are known in the art and can be used in accordance with the present invention for producing a population of lipid nanoparticles of particular size ranges, for example, sonication or homogenization.

[0158] The size of nanoparticles can be measured using a variety of techniques such as dynamic light scattering, size exclusion chromatography, nuclear magnetic resonance spectroscopy, and microscopy. The particle size distribution of LNPs can be controlled using manufacturing methods such as extrusion, sonication, and homogenization; more recently, microfluidic methods have been successfully used for LNP manufacture and size control. The size of the lipid nanoparticles may also be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421A 150 (1981).

[0159] In some embodiments of the method, the polydispersity index of the lipid nanoparticles is less than about 0.3, or less than about 0.2.

[0160] In some embodiments of the method, the zeta potential of the lipid nanoparticles is from about -40 mV to about 40 mV, or from about -10 mV to about 10 mV.

[0161] Preferably, the cells are incubated with a concentration of LNPs of 0.1-5 pg / 500.000 cells, more preferably of 0.5-4 pg / 500.000 cells, most preferably of 1-3 pg / 500.000 cells.

[0162] The present invention is further directed to the engineered cells obtained by the method of the invention and to pharmaceutical formulations comprising the population of engineered cells of the invention and pharmaceutically acceptable excipients. The term "pharmaceutically acceptable excipient" refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any conventional type that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient. A pharmaceutically acceptable excipient is essentially nontoxic to recipients at the employed dosages and concentrations and is compatible with other ingredients of the formulation. The number and the nature of the pharmaceutically acceptable excipients depend on the desired administration form.

[0163] Preferably the method of the invention further comprises the steps of:

[0164] - collecting the genetically modified cells, and optionally

[0165] - resuspending the cells in a freezing medium.

[0166] Preferably, after collection, the genetically modified cells are then washed and resuspended, more preferably at a concentration of 2.5-10 x 106cells / ml, in a minimum volume of freezing medium (e g., 20 ml), and cryopreserved under vapor of liquid nitrogen in cryobags.

[0167] The cells can be frozen until use.

[0168] Preferably the cells are autologous cell, i.e., cells obtained from a subject, to which the cells are meant to be reinfused, once genetically modified.

[0169] Preferably, the invention provides a population of engineered cells, more preferably T cells or HSPCs cells, obtained by the method of the invention, or a pharmaceutical formulation thereof for use in the treatment or prevention of a disease.

[0170] The population of engineered cells obtained by the method of the invention may be useful in the treatment of the disorders listed in WO 1998 / 005635. For ease of reference, part of that list is now provided: cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, hemorrhage, coagulation and acute phase response, cachexia, anorexia, acute infection, HIV infection, shock states, graft-versus-host reactions, autoimmune disease, reperfusion injury, meningitis, migraine and aspirin-dependent anti-thrombosis; tumor growth, invasion and spread, angiogenesis, metastases, malignant, ascites and malignant pleural effusion; cerebral ischemia, ischemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer’s disease, atherosclerosis, stroke, vasculitis, Crohn’s disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; corneal ulceration, retinopathy and surgical wound healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis or endosclerosis.

[0171] In addition, or in the alternative, the population of engineered cells obtained by the method of the invention may be useful in the treatment of a disorders listed in WO 1998 / 007859. For ease of reference, part of that list is now provided: cytokine and cell proliferation / differentiation activity; immunosuppressant or immunostimulant activity (e.g. for treating immune deficiency, including infection with human immune deficiency virus; regulation of lymphocyte growth; treating cancer and many autoimmune diseases, and to prevent transplant rejection or induce tumor immunity); regulation of hematopoiesis, e.g. treatment of myeloid or lymphoid diseases; promoting growth of bone, cartilage, tendon, ligament and nerve tissue, e.g. for healing wounds, treatment of burns, ulcers and periodontal disease and neurodegeneration; inhibition or activation of follicle-stimulating hormone (modulation of fertility); chemotactic / chemokinetic activity (e.g. for mobilizing specific cell types to sites of injury or infection); hemostatic and thrombolytic activity (e.g. for treating hemophilia and stroke); anti-inflammatory activity (for treating e.g. septic shock or Crohn’s disease); as antimicrobials; modulators of e.g. metabolism or behavior; as analgesics; treating specific deficiency disorders; in treatment of e.g. psoriasis, in human or veterinary medicine.

[0172] In addition, or in the alternative, the invention may be useful in the treatment of the disorders listed in WO 1998 / 009985. For ease of reference, part of that list is now provided: macrophage inhibitory and / or T cell inhibitory activity and thus, anti-inflammatory activity; anti-immune activity, i.e. inhibitory effects against a cellular and / or humoral immune response, including a response not associated with inflammation; inhibit the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, as well as up-regulated of receptor expression in T cells; inhibit unwanted immune reaction and inflammation including arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto-rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia, preeclampsia and other immune and / or inflammatory-related gynecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreo-retinopathies, acute ischemic optic neuropathy, excessive scarring, e g. following glaucoma filtration operation, immune and / or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and / or inflammation suppression would be beneficial, Parkinson’s disease, complication and / or side effects from treatment of Parkinson’s disease, AIDS-related dementia complex HIV-related encephalopathy, Devic’s disease, Sydenham chorea, Alzheimer’s disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumor cerebri, Down’s Syndrome, Huntington’s disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and / or side effects, inflammatory and / or immune complications and side effects of gene therapy, e g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and / or cellular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukemia, by reducing the amount of monocytes or lymphocytes, for the prevention and / or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue.

[0173] The present invention is then directed also to a population of genetically modified cells or the pharmaceutical formulation, obtained by the method of the invention, for use as a medicament, in the treatment or prevention of a disease, more preferably of a disease selected from cancer, an immune disorder, abacterial or viral infection, a genetic disease, a blood disease, a lysosomal storage disease, P-thalassemia, Fanconi anemia, bone marrow failure disease, sickle cell disease, osteopetrosis, chronic granulomatous disease, metachromatic leukodystrophy, mucopolysaccharidosis.

[0174] In preferred embodiments, the engineered cells are administered as part of an autologous stem cell transplant procedure.

[0175] In other preferred embodiments, the engineered cells are administered as part of an allogeneic stem cell transplant procedure.

[0176] Engineered T cells obtained by the method of the invention can be used in methods of ex vivo cell therapies that are based on infusion of modified T cells into patients, including anti-cancer strategies (such as using engineered CAR-T cells) and approaches based on infusion of universal donor T cells.

[0177] It should be understood that all the possible combinations of the preferred aspects of the present invention are also described, and therefore similarly preferred.

[0178] Examples of preferred embodiments of the present invention and analyses of their efficacy are provided below for illustrative and non-limiting purposes.

[0179] EXAMPLES

[0180] MA TERIALS AND METHODS

[0181] The material and methods described hereafter have been used in the Examples that follow. Gene editing

[0182] AAV6 production and sgRNA sequences were previously reported (Vavassori V et al. EMBO Mol. Med. 2021;13(3): el3545; Giulia Schiroli A, et al. Stem Cell. 2019; 24:551-565. e8; Gaudelli NM, et al. Nat. Biotechnol. 2020 387. 2020;38(7): 892-900).

[0183] RNPs were assembled by incubating at 1 :2 or 1 : 1 5 molar ratio 5pCas9 protein (Aldevron) with sgRNA (Synthego). CRISPR / Cas9 RNA was prepared by mixing CleanCap® Cas9 mRNA (5moU, Trilink) with sgRNA at 2.49: 1 mass ratio.

[0184] For electroporation, 5xl05 / 106T cells or 2xl05 / 7.5xl05CB / mPB-HSPCs for each condition were treated after 3 days of stimulation, as previously reported (Vavassori V et al. EMBO Mol. Med. 2021; 13(3): el3545; Ferrari S et al. Nat. Biotechnol. 2020; 1-11) and as detailed hereafter. 3pg GSE56 mRNA (Giulia Schiroli A, et al. Stem Cell. 2019; 24:551-565. e8; Gaudelli NM, et al. Nat. Biotechnol. 2020387. 2020;38(7):892-900) were electroporated along with RNP where indicated.

[0185] LNPs were formulated and quantified as detailed hereafter. Empty LNPs were prepared by substituting RNA with water.

[0186] LNPs were stored at 4°C for <1 week.

[0187] After 3 days of stimulation, 5xl05T cells or 3xl05CB / mPB-HSPCs for each condition were seeded at a concentration of 5xlO5cells / ml. 0.1 ug / ml recombinant human ApoE was added to the medium, cells were resuspended and incubated with different doses of CRISPR / Cas9 RNA- LNPs or the same volume of Empty LNPs.

[0188] AAV6 transduction was performed 0-2 hours before LNPs addition at dose of 5xl04 / 2xl04vg / cell, unless otherwise specified. 24 hours later, cells were washed with DPBS and reseeded. IDLV transduction was performed 10 hours before LNP transfection at MOI 40, with or without LentiBoost addition, as indicated. 24 hours later, cells were washed with DPBS and reseeded. Molecular analyses

[0189] Deep sequencing, ddPCR and T7 assay for HDR / NHEJ assessment were performed on purified DNA. For gene expression, RNA was extracted and 2ng of cDNA were used for CDKN1A (Hs00355782_ml, Life Technologies) and APOBEC3H (Hs00419665_ml) ddPCR as previously reported (Ferrari S, et al. Nat. Biotechnol. 2020; 1-11).

[0190] RNA-seq was performed using DESeq2. Proteomic analysis was performed on 3 healthy donors (HD), as described hereafter.

[0191] Primary cells and functional assays Peripheral blood mononuclear cells (PBMCs) were purified from buffy coats in accordance with the Declaration of Helsinki, as anonymized residues of blood donations, used upon signature of specific institutional informed consent for blood product donation CD3+ or CD4+ T cells were isolated using Pan T cell isolation kit or CD4 T cells isolation kit (Miltenyi Biotech), stimulated and cultured as previously described (Vavassori et al. EMBO Mol Med 2021; 13: el3545).

[0192] For the GMP -compliant T cell culture protocol, CD3 or CD4 T cells were stimulated using 60 pl / 1.5xl06cells of T Cell TransAct™ reagent (Miltenyi Biotec) and cultured in X-VIVO 15 medium (Lonza) supplemented with 0.5% of Human Serum Albumin (Baxalta), IL-7 (lOOUI / ml, Miltenyi Biotec) and IL- 15 (200UI / ml, Miltenyi Biotec), 1% penicillin / streptomycin (Lonza). Low doses of IL-2 (50UI / ml, Miltenyi Biotec) were supplemented into culture medium 3 days after seeding. 5% Human Serum (PAN Biotech) was added 24 hours after LNP wash (24 hrs after LNP incubation).

[0193] Human CB or mPB CD34+ HSPCs were purchased from Stem Express or in-house purified from Mobilized Leukopak (AllCells), respectively and cultured as previously described (Ferrari S, et al. Front. Genome Ed. 2021; 0:99). Briefly, cells were seeded at the concentration of 5xlO5cells / ml in serum-free StemSpan SFEM (STEMCELL Technologies) supplemented with penicillin (100 lU / ml), streptomycin (100 pg / ml), 2% glutamine, human early-acting cytokines (SCF 100 ng / ml, Flt3-L 100 ng / ml, TPO 20 ng / ml, and IL-6 20 ng / ml; Peprotech), 1 pM SR- 1 (Biovision), 50 nMUM171 (STEMCell Technologies) and 10 pM dmPGE2 (Cayman) added only right-after thawing.

[0194] G-CSF or G-CSF + Plerixafor mPB CD34+ HSPCs were purified in house with the CliniMACS CD34 Reagent System (Miltenyi Biotec) from Mobilized Leukopak (AllCells) following the manufacturer’s instructions. HSPCs were seeded at the concentration of 5xl05cells / ml in serum-free StemSpan SFEM supplemented with penicillin (100 lU / ml), streptomycin (100 pg / ml), 2% glutamine, human early-acting cytokines (SCF 300 ng / ml, Flt3-L 300 ng / ml, TPO 100 ng / ml; Peprotech), 1 pM SR-1 (Biovision), 35 nM UM171 (STEMCell Technologies) and 10 pM dmPGE2 (Cayman) added only right-after thawing. Clinically compliant conditions included CellGenix® GMP SCGM culture medium, (SCF 100 ng / ml, Flt3-L 100 ng / ml, TPO 50 ng / ml, IL3 50 ng / ml, IL6 50 ng / ml; CellGenix) 1 pM SR-1 (Biovision), 35 nM UM171 (STEMCell Technologies).

[0195] Colony-forming-unit HSPCs assay was performed as previously described (Ferrari S, et al. Front. Genome Ed. 2021;0:9). For polyclonal T cell stimulation, T cells were removed from PBMCs isolated from three healthy donors using CD3 microbeads (Miltenyi Biotech). T cell depleted PBMCs were equally mixed and irradiated (60Gly). At the end of gene editing procedure edited T cells and controls were harvested and co-cultured in triplicate (105cells / well, ratio 1:1) for 72 hrs in X-VIVO 15 medium (Lonza), supplemented with 5% human serum and lOOU / ml penicillin / streptomycin with irradiated T cell depleted feeder mix ± anti human CD3 mAh (clone OKT3, Ipg / mL). Proliferation was quantified by thymidine incorporation adding to cell in culture 1 mCi / well [3H]thymidine for additional 16 hrs of culture.

[0196] Levels of IFN-y released in the medium were determined by ELISA according to the manufacturer’s instructions (BD Biosciences) at the indicated time point.

[0197] Allogeneic reactivity determination by Mixed leucocyte reaction (MLR). At the end of gene editing procedure edited T cells and controls were harvested and co-cultured in triplicate (105cells / well ratio 1:1) for 72, 96, and 120 hrs with irradiated (60Gly) T cell depleted PBMCs of 3 independent healthy donors. Proliferation was quantified by thymidine incorporation. After the indicated time of co-culture, cells were pulsed for 16 hrs with 1 mCi / well [3H]thymidine. Preparation ofLNPs

[0198] A commercial kit was used for LNPs preparation, according to manufacturer’s instructions. Briefly, a single reaction of mRNA working solution was prepared by mixing 11 pg of CRISPR / Cas9 mRNA, prepared as described below, or GFP mRNA (5moU - Trilink) with 3.52 pl of Formulation Buffer 10X and filled up to 35.2 pl with sterile water. CRISPR / Cas9 mRNA was prepared by mixing CleanCap® Cas9 mRNA (5moU - Trilink) with sgRNA (Synthego) at a Cas9 / sgRNA ratio of 2.49: 1. Empty LNPs were prepared by substituting CRISPR / Cas9 mRNA with sterile water. HDR enhancers mRNA were produced by TebuBio and either formulated independently or co-formulated with CRISPR / Cas9 CleanCap® Cas9 mRNA (5moU - Trilink) and sgRNA (Synthego) at 1 / 3 ratio. ABE8.20-m mRNA was produced by TebuBio and formulated with sgRNA (Synthego) at a ABE8.20-m / sgRNA ratio of 2.49: 1. The largest well of the cartridge was then filled with 48 pl of Dilution Buffer, the middle well with 32 pl of mRNA working solution and the smallest well with 16 pl of Lipid Mix. The cartridge was then inserted into the instrument and LNPs formulated with Setting 3. Once the formulation step was completed, the formulated mRNA-LNPs were moved into 96 pl of Dilution Buffer and stored at 4°C for one week at most.

[0199] Quantification of encapsulated mRNA was performed using Quant-it™ RiboGreen RNA Assay Kit (Thermo Fisher Scientific), following manufacturer’s instructions. Size and poly dispersity index were measured by analyzing 40 pl of LNPs diluted in 900 pl of PBS using the ZetaSizer (Malvern) instrument and default parameters.

[0200] Gene editing of primary cells using electroporation

[0201] AAV6 production and sgRNA sequences were previously reported (Vavassori V et al. EMBO Mol. Med. 2021; 13(3): el3545; Ferrari S et al. Nat. Biotechnol. 1-11, 2020; Gaudelli NM et al. Nat Biotechnol. 2020 Jul;38(7):892-900). RNPs were assembled by incubating at 1:2 or 1 : 1.5 molar ratio ,S / ?Cas9 protein (Aldevron) with sgRNA (Synthego). CRISPR / Cas9 RNA was prepared by mixing CleanCap® Cas9 mRNA (5moU, Trilink) with sgRNA at 2.49: 1 mass ratio. Primary T cells: After 3 days of stimulation, 5xl05 / 106cells derived from healthy donors for each condition were washed with ten volumes of DPBS and electroporated with 25 pmol or 50 pmol of RNP or with different doses of CRISPR / Cas9 mRNA, prepared as described above (P3 Primary Cell 4D-Nucleofector X Kit, programs DS-130; Lonza). AAV6 transduction was performed 15 minutes after electroporation at a dose of 5xl04vg / cell. 3 pg of GSE56 mRNA was transfected along with RNP when indicated. IDLV transduction was performed 10 hours before electroporation at MOI 40, with or without LentiBoost addition, as indicated.

[0202] CB- / mPB-derived HSPCs: After 3 days of stimulation, 2xl05 / 7.5xl05cells for each condition were washed with ten volumes of DPBS and electroporated with 25 pmol or 50 pmol of RNP or with different doses of CRISPR / Cas9 mRNA, prepared as described above (P3 Primary Cell 4D-Nucleofector X Kit, program EO-100; Lonza). AAV6 transduction was performed 15 minutes after electroporation procedure at a dose of 2xl04vg / cell.

[0203] Flow cytometry

[0204] Immunophenotypic and apoptosis analyses were performed as described by Ferrari S, et al. in Front. Genome Ed. 2021; 0:9 (Table 1). CD90high HSC Sorting was performed with MACSQuant® Tyto® Cell Sorter (Miltenyi Biotech).

[0205] Mitochondrial membrane potential analysis was performed 2 and 24 hours after editing using MitoTracker® Green FM and Red FM (Invitrogen), according to manufacturer’s instructions. MitoTracker® Red MFI was normalized on MitoTracker® Green MFI for analysis.

[0206]

[0207] Table 1. List of antibodies used in this study. HSPC xenotransplantation

[0208] Procedures were performed upon approval of hosting institution and Italian authorities (IACUC 1206). Female NOD.Cg-PrkdcscidI12rgtmlWjl / SzJ mice (The Jackson Laboratory) were held in specific pathogen-free conditions. For xenotransplantation of CB and mPB CD34+ HSPCs, the outgrowths of 5xl04and 7.5xl05 / 106HSPCs, respectively, seeded at the start of the culture (time 0, tO) were injected intravenously 24 h after editing into 8 weeks old female NSG mice,

[0209] 4 hours after sublethal irradiation (150-200 cGy).

[0210] Statistical analyses

[0211] The number of biologically independent samples is indicated by “n”. Data were summarized as median with interquartile range (IQR), unless otherwise specified. Two-tail tests were performed. Standard comparisons of numerical variables between two groups were evaluated either with Mann-Whitney or Wilcoxon’s paired test, whilst Kruskal-Wallis or with Friedman test, followed by a post hoc analysis with Dunn’s test with Bonferroni’s correction, were used in case of three or more groups. Significance threshold was 0.05. “ns” means not significant. Analyses were performed using GraphPad Prism v.9.3.1 (GraphPad).

[0212] Data and code availability

[0213] Sequencing data have been deposited in Gene Expression Omnibus (GSE215250). Mass- spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol Y, et al. Nucleic Acids Res. 2022;50(Dl): D543-D552) partner repository (PXD037529). Scripts for RNA-Seq and proteomic analysis are available at http: / / www.bioinfotiget.it / gitlab / custom / vavassori_lnp2022.

[0214] Culturing and gene editing of CD4+ T-cells with IDLV transduction of donor template

[0215] Small (l-2xl06cells at day 2) and medium scale (5-10xl06cells at day 2) experiments were carried out, wherein CD4+ T-cells were isolated from buffy coats of healthy donors with StraightFrom® Buffy Coat CD4 Microbeads or from patients’ peripheral blood samples with StraightFrom® Whole Blood CD4 Microbeads human (both Miltenyi Biotec).

[0216] CD4+ T-cells were cultured in X-Vivo 15 (Lonza) supplemented with 0.5% Human Serum Albumin (HSA, Baxalta), 1% Pen / Strep (Lonza), 100 lU / ml IL7 and 200 lU / ml IL15 (both from Miltenyi Biotec) and stimulated with T Cell TransAct (Miltenyi Biotec) (40 microl / 106cells) at 2xl06cells / ml.

[0217] On day 2, unless stated otherwise, cells were transduced at IxlO6cells / ml with IDLV delivering a donor template encoding for a splice acceptor sequence and the cDNA of CD40LG exons 2-

[0218] 5 (CD40LG-IRES LNGFR-IDLV), at MOI 40. The corrective donor template includes a low affinity nerve growth factor receptor (LNGFR) selector, by design expressed only upon correct integration of the corrective cassette, through an Internal Ribosome Entry Sites (IRES) sequence.

[0219] After 8±1 hours from transduction, cells were either electroporated with SpyFi™ high fidelity (HiFi) Cas9 (Aldevron) complexed with a single guide RNA (gRNA) targeting the first intron of CD40LG or contacted with LNPs in a serum-free culture medium, said LNPs comprising Cas9 and gRNA targeting the first intron of CD40LG. Electroporation was performed with Maxcyte Process Assembly OC-25x3 or OC- 100x2 using MaxCyte GTx Electroporator.

[0220] After electroporation or LNPs treatment, cells were cultured in X-Vivo 15 (Lonza) supplemented with 5% Human Serum (HS, Pan Biotech), 1% Pen / Strep (Lonza), 100 lU / ml IL7, 200 lU / ml IL15 and 50 lU / ml IL2 (all from Miltenyi Biotec). On day 6, unless stated otherwise, cells were enriched by custom-made immunomagnetic CD271 Microbeads (Miltenyi Biotec) and subsequently expanded until day 13-14 in the same culture medium. Cells were then frozen in saline solution supplemented with 7% HSA, 5% DMSO (Sigma) with KryoPlaner560-16 Software version v5.26 (Planer Limited, Sunbury-On-Thames, UK) and stored in nitrogen vapors.

[0221] Multiwell plates (24w) were used for small scale experiments.

[0222] EXAMPLE 1

[0223] Impact of electroporation on human CD4+ T cells

[0224] A multi-parametric analysis was performed investigating the impact of each step of gene editing procedure on human CD4+ T cells from three healthy donors (HD), using previously optimized culture / editing protocol and reagents targeting CD40LG (Vavassori V, et al. EMBO Mol. Med. 2021;13(3):el3545). Untreated cells (UT), mock electroporated cells (mock electro), cells electroporated with RNP (RNP), cells electroporated with RNP and AAV6 donor (RNP+AAV6), and AAV6-transduced cells (AAV6 only, no electroporation) were compared (Figure 1A). Editing efficiency was robust, reaching up to -75% NHEJ and -50% HDR, according to treatment, preserving a substantial proportion of the clinically relevant T stem memory (TSCM) and central memory (CM) cells (Figure IB). Strikingly, a major increase of apoptotic / necrotic cells after mock electroporation was found, reaching up to 50% of the cells and only slightly worsened when introducing the editing components. Cell death in AAV6- transduced cells was nearly comparable to UT (Figure 1C). Concordantly, electroporated cells showed an initial delay, up to halted cell growth upon addition of editing components, which recovered overtime (Figure ID). Mock electro, RNP and RNP+AAV6 samples showed similar paterns of p53 / apoptotic / inflammatory genes upregulation, highlighting that addition of editing components increased cumulatively, but limitedly, the response induced by electroporation per se.

[0225] EXAMPLE 2

[0226] LNP-mediated CRISPR / Cas9 RNA delivery improves T cell tolerance to gene editing

[0227] LNP -based delivery of the Cas9 mRNA and sgRNA targeting CD40LG was explored (Figure 2A), using a commercial formulation for ex vivo T cells transfection, having median size around 130 nm, poly dispersity index <0.3 (Danaei M, et al. Pharmaceutics. 2018;10(2)) and excellent transfection efficiency.

[0228] Presence of serum in culture media nearly abolished editing efficiency (Figure 6A), likely due to LDL-induced down-regulation and competition for binding to its receptor (LDLR) (Figure 6B). LDLR expression and editing efficiency were similar using different T cell activation reagents (Figure 6A-B).

[0229] Surprisingly, the serum-free culture protocol, according to the invention, wherein cells are maintained in serum-free medium before and during editing, yielded robust cell growth and increased LDLR expression (Figure 6B-C). This was particularly evident in the conditions wherein a GMP-compliant culture protocol was used. LNP transfection was less efficient per RNA mass at lower doses but reached similar NHEJ efficiency at the highest ones compared to electroporation of RNA / RNP (Figure 6D). Strikingly, LNP transfection did not increase the percentage of apoptotic / necrotic cells early after treatment when compared to UT (Figure 6E). However, increasing growth arrest and cell death was observed over time in cells treated with LNPs (Figure 6F).

[0230] Surprisingly, washing out LNPs 24 hours after incubation prevented toxicity, without affecting NHEJ efficiency (Figure 6D-F).

[0231] Using this optimized protocol, a multiparametric side-by-side comparison was performed of LNP- and RNA / RNP electroporation- mediated editing in CD4+ T cells from six HD (Figure 2A).

[0232] LNP-editing was carried out according to the method of the invention, including the serum-free incubation of LNPs and wash out of LNPs after incubation.

[0233] Robust NHEJ editing (<80%) was achieved by all treatments (Figure 2B) with comparable distribution of indels size across the different delivery modalities at matched editing efficiencies. The increased apoptosis / necrosis in electroporated samples was nearly absent in LNP -treated ones (Figure 2C). Measurement of the mitochondrial potential showed depolarization immediately after electroporation followed by sustained hyperpolarization (Figure 2D), the earliest change associated to apoptosis. Conversely, LNP -treated cells, according to the method of the invention, did not show altered mitochondrial potential (Figure 2D) and showed minimally decreased growth, even short term after editing (Figure 2E). Consequently, the early yield of NHEJ-edited cells doubled with LNP treatment (Figure 2F), although electroporated samples recovered in the long term with faster kinetics than in earlier studies, due to improved culture conditions (Figure 6C). None of the treatments induced significant phenotypic cell skewing (Figure 2G).

[0234] LNP transfection did not impact viability and only minimally decreased growth of CD3+ T cells edited at another locus (B2M exon 2), as opposed to electroporation, while preserving TSCM and CM cells and promoting efficient editing (75% biallelic knockout, measured by flow cytometry) without fostering longer deletions (Figure 2H-K,). Because of the lower impact on cell growth, the early yield of edited cells was more than doubled compared to electroporation (Figure 2L).

[0235] Next, it was investigated whether LNP delivery could be applied for HDR-based editing, targeting CD40LG with a therapeutic cassette containing a reporter. LNPs were less efficient than electroporation in promoting HDR at all doses tested (Figure 6G), likely due to competition for entering cells or interference with leaving endosomes. None of the tested permutations of LNP / AAV6 delivery improved HDR (Figure 6H-I). A multiparametric comparison of LNP- based and RNA / RNP electroporation was performed in CD4+ T cells from 8 HD (Figure 2A) for CD40LG editing. Whereas HDR efficiency was again one third lower for LNP-treated cells (Figure 2M-N), there was nearly absent increase of apoptotic / necrotic cells, mirrored by normal mitochondrial potential, as opposed to electroporated cells (Figure 2D, O). Consistently, LNP- based editing only minimally delayed growth, even short term after editing (Figure 2P). As a result, the yield of HDR-edited cells early after editing was similar or even increased for LNP- treated samples (Figure 2Q). None of the treatments induced significant phenotypic cell skewing (Figure 2R). Similar results were obtained when targeting by HDR a GFP expression cassette into AAVS1 in CD3+ cells (Figure 2S-W), confirming the advantages of the use of LNPs and removal of the same after incubation, in accordance with the conditions of the method of the invention.

[0236] EXAMPLE 3

[0237] LNP-mediated cholesterol overloading triggers an adverse response, overcome after particle withdrawal To characterize the impact of LNPs in T cells, transcriptomic analysis was performed 24 hours after CD40LG editing in CD4+ T cells.

[0238] LNP-mediated gene editing was carried out according to the method of the invention.

[0239] As controls, cells untreated, mock electroporated or treated with LNPs formulated without RNA (Empty LNPs) were employed 5A-E). First the Mock electro-UT comparison was carried out. Again, over-representation analysis (ORA) highlighted significant upregulation of genes belonging to apoptosis, p53 pathway and inflammatory categories and downregulation of cell cycle target genes (not shown). IFN responses were negatively enriched in electroporated cells, as opposed to previous findings. Consistently with the faster recovery of cell growth, downregulation of metabolism-related genes was no longer observed in electroporated samples, confirming the improvement in culture conditions.

[0240] When comparing cells treated with empty or RNA-containing LNPs with UT, ORA highlighted similar upregulation of p53, apoptosis and inflammatory genes, comparable to electroporated samples. However, distinct sets of genes belonging to p53 and apoptosis categories subclustered according to their expression upon different treatments. While electroporation enriched for positive regulation of apoptosis, LNPs enriched for genes implicated in its negative regulation, concordantly with the lower cell death. IFN pathways were positively enriched in Empty LNPs / LNPs vs UT, as opposed to electroporated ones. Moreover, Empty LNPs / LNPs samples showed downregulation of genes belonging to cholesterol homeostasis (e.g., LDLR, SQLE and SREBF2) when compared to UT and electroporated samples, likely in response to the cellular loading with exogenous lipids. In contrast to the preserved proliferation, Empty LNPs / LNPs samples showed downregulation of cell cycle related genes compared to UT and electroporated samples. It has been reported that cholesterol homeostasis in proliferating T cells is maintained through opposite regulation of SREBF2 and LXR transcriptional programs. While upon T cell activation SREBF2 transcribes genes involved in cholesterol biosynthesis and uptake, excess cholesterol activates LXR that transcribes target genes such as ABCA1 and ABCG1, which are membrane transporters mediating cholesterol efflux. Enforced expression o ABCGl engages a metabolic checkpoint blocking proliferation. Empty LNPs / LNPs samples showed downregulation of SREBF2 and its target genes and upregulation of ABCAI and ABCG1 compared to other conditions, consistently with cholesterol loading from LNPs and possibly explaining the observed impact on cell cycle. Moreover, induction of interferon response has been reported upon SREBF2 downregulation, possibly explaining upregulation of genes in these categories in LNP-treated vs electroporated cells. These adverse cellular responses may explain the irreversible growth arrest and toxicity observed when LNPs were left in the medium (Figure 6F). Upon long-term culture, LNP-edited cells did not show signs of exhaustion by cell surface analysis, with negligible proportions of cells expressing multiple inhibitory receptors, and responded to polyclonal re-stimulation and / or Mixed Leucocyte Reaction with similar to higher IFN-y production and proliferation levels compared to UT or electroporated cells (Figure 3 A-B), supporting preserved functionality as the other treatments, when LNPs are withdrawn after incubation

[0241] EXAMPLE 4

[0242] LNP-mediated CRISPR / Cas9 delivery improves tolerance of human HSPC to ex vivo gene disruption

[0243] LNP-based gene editing in human HSPCs was investigated, comparing LNPs and electroporation for B2M disruption in mobilized peripheral blood (mPB) HSPCs, using previously optimized culture conditions (Giulia Schiroli A, et al. Stem Cell. 2019; 24:551- 565 e8) that showed robust LDLR expression in all progenitor subsets (Figure 4Q). LNP -treated cells, according to the method of the invention, showed higher / / 2 / V / -KO efficiency compared to electroporation across all subpopulations at matched RNA doses, reaching -50% in the primitive compartment (CD34+CD133+CD90+). Shortening LNP incubation decreased B2M- KO (Figure 4R). Since electroporation of Cas9 RNP is the current state-of-the-art for HSPC editing, LNP- and RNP -mediated editing was compared in six mPB-HSPC donors (Figure 4A). Despite RNA-LNPs reaching lower editing efficiency than RNP at the highest doses, the yield of B2M-K0 cells was comparable for both treatments (Figure 4B-C), given the improved viability and growth of LNP -treated cells (Figure 4D-E). None of the treatments induced significant phenotypic cell skewing (Figure 4F). A cumulative impact of electroporation and nuclease-induced DNA DSBs on HSPC clonogenic potential was observed, which instead was not affected by LNPs (Figure 4G). Consistently, p21 and APOBEC3H induction 24 hours after editing was lower for LNP samples (Figure 4H), reflecting an exclusive input on the p53 pathway from nuclease-induced DSBs, rather than cumulative inputs from DSBs and electroporation. Similar results were obtained when treating HSPCs from cord blood or when editing mPB HSPCs in the exon 1 of B2M using a more efficient sgRNA (Supplemental (not shown). Of note, nuclease delivery by LNPs yielded a similar proportion of NHEJ-edited alleles and did not increase the proportion of on-target longer deletions at matched editing efficiencies compared to the other delivery modalities.

[0244] Whole transcriptomic analysis 24 hours after AAVS1 editing with LNPs or electroporation of RNA / RNP in mPB-HSPCs, using as controls cells untreated, mock electroporated or treated with empty LNPs, showed that LNP transfection was as efficient as RNP electroporation in promoting NHEJ at AAVS J and slightly more efficient than RNA electroporation at matching doses. ORA highlighted significant upregulation of genes belonging to categories involved in DDR, apoptosis and inflammation when comparing all electroporated samples with UT, with increasing response in the RNP and RNA samples. A significant enrichment was observed, among downregulated DEGs, of categories involved in cell cycle, oxidative phosphorylation and fatty-acid metabolism / adipogenesis. Conversely, Empty LNPs did not activate DDR and inflammation, which were instead triggered by LNPs carrying RNA, thus by editing components and DSBs, when compared to UT. The same comparison showed downregulation of cholesterol homeostasis and cell cycle related genes, as already observed in T cells. Interestingly, distinct sets of p53-related genes subclustered according to expression upon treatment, with electroporation enriching for DDR genes different from those specifically induced by nuclease exposure, and LNP vehicle enriching for a smaller subset involved in apoptosis regulation.

[0245] Immunodeficient mice were then transplanted with the outgrowth of 7.5xl05mPB-HSPCs seeded at the start of the culture (time 0, tO), edited at B2M with LNPs or electroporation of either RNA / RNP and similar engraftment of human cells found for all treatments, as monitored longitudinally in PB and hematopoietic organs at the end of experiment (Figure 41- J). Gene editing efficiencies, as revealed by B2M- O, were also similar among groups, with slight decrease from early to later times (Figure 4K-L). A similar experiment was then performed with CB-HSPCs and transplanted 5xl04outgrowth of tO seeded cells edited either at B2M or A A VS1 by LNPs or RNP electroporation. LNP-treated cells showed similar engraftment early after transplant but higher human graft size at later times in PB, spleen and BM in one of two experiments (Figure 4M-N, Figure 7 A-B). B2M and AAVS J editing efficiencies in human xenografts were confirmed by deep sequencing with similar ranges reached by either RNA delivery platform (Figure 4O-P, Figure 7 C).

[0246] EXAMPLE 5

[0247] LNP-mediated CRISPR / Cas delivery supports HDR editing of HSPCs

[0248] It was then investigated whether LNP delivery and removal of the same after incubation, in accordance with the method of the invention could be suitable for HDR-based editing, targeting AAVS1 with a GFP cassette in HSPCs. LNPs outperformed electroporation at matched low RNA doses and reached similar or better HDR at maximal doses across all subpopulations. LNP- and RNP-mediated editing was compared in mPB-HSPCs from six HD (Figure 5A) and found similar HDR editing in CD90+ cells (Figure 5B). However, since electroporated HSPCs showed higher toxicity and delayed growth, the yield of CD90+GFP+ cells increased for LNP- treated samples (Figure 5C-E). None of the treatments changed subset composition compared to UT (Figure 5F). Impaired clonogenic potential and robust p21 and APOBEC3H induction was observed in cells edited with electroporation or LNPs, where the dominant AAV6 effect masked the advantage provided by the latter platform (Figure 5G-H). Similar results were obtained when treating CB-HSPCs.

[0249] Finally, 106outgrowths of tO seeded mPB-HSPCs edited at AAVS1 by LNPs or electroporation were transplanted in mice. PB, BM and spleen analyses revealed multi-lineage long-term engraftment for both treatments, with higher graft size for the LNP group in one of two experiments performed with different pools of HD (Figure 5I-L). The percentage of GFP+ cells in organs was comparable between the conditions tested and showed a slight decrease at longer times (Figure 5M-P).

[0250] EXAMPLE 6

[0251] CD4+ cells were isolated from buffy coats of healthy donors using magnetic columns, then activated.

[0252] After 2-3 days, they were edited in CD40L by electroporation of Cas9 RNA / RNP or by delivery of Cas9 with LNPs, followed by removal of the same according to the method of the invention. IDLV was used to deliver the CD40LG donor template.

[0253] On day 6 the cell product was enriched for LNGFR+ cells, with high purity and a good recovery. Overall, CD4+ purity was maintained until the end of the process with no expansion of CD8+ T cells.

[0254] Editing efficiency was higher by electroporation (Figure 8); however, editing with LNPs provided higher survival rates and increased vitality of cells.

[0255] EXAMPLE 7

[0256] The optimized LNP -mediated editing protocol of the invention, comprising incubating cells with the LNPs in a serum-free culture medium and including removal of the LNPs after incubation, was used in a clinically compliant protocol, using an expansion medium for mPB- derived HSPCs (Figure 9 A). Regardless of the LNP doses used to deliver the editing reagents, an improved HDR efficiency in the AAVS1 locus was observed with the clinically compliant protocol, as indicated by the percentage of GFP expression measured by flow cytometry (Figure 9 B). Furthermore, commercial LNP formulations optimized for HSC delivery as well as LNP formulation optimized for T cell delivery were used in four mPB-derived HSPC donors. NHEJ efficiency was measured through the lack of B2M expression (Figure 9 C) and HDR efficiency was measured by GFP expression (Figure 9 D), in the most primitive compartment.

[0257] EXAMPLE 8

[0258] To further enhance the HDR editing protocol of the invention, the editing reagents were codelivered in HSPCs by LNPs, together with an editing enhancer (GSE56 or GSE56 / Ad5- E4orf6 / 7 were used indifferently) (Figure 10 A). In accordance with the method of the invention, LNPs were removed after incubation.

[0259] The use of editing enhancers further improved HDR efficiency, achieving up to 70% GFP expression in the most primitive compartment (Figure 10 B) and remarkably mitigated detrimental p53 responses, as shown by the reduction of p21 and APOBEC3H induction (Figures 10 C-D).

[0260] EXAMPLE 9

[0261] Finally, the feasibility of the method of the invention for delivering base editors to human HSPCs was assessed. To do this, LNP-mediated transfection, followed by removal of LNPS after incubation, was compared to a previously optimized protocol based on electroporation (Fiumara M. et al, 2023- Nat Biotechnol) to deliver an adenine base editor (ABE8.20-m) mRNA and a sgRNA targeting the B2M gene (Figure 11 A). Moreover, different timings for base editing were compared: 12 hours versus 3 days of culture after thawing. Indeed, whereas a longer protocol promotes metabolic activation and cell cycle progression, a shorter protocol may better preserve stem cell phenotype.

[0262] By assessing the lack of B2M expression within the most primitive compartment, the LNP- based protocol was found to be superior to the electroporation-based method, achieving 90% efficiency at both 12 hours and 3 days of culture (Figure 11 B).

[0263] EXAMPLE 10

[0264] Next, the feasibility of the method of the invention for delivering ABE, in the context of primitive HSC selection and expansion in a clinically compliant process, was assessed (Figure 12A). CD90hlghprimitive HSCs were enriched using a GMP-compliant cell sorter after 36 hours of pre-stimulation and transfected at 48 hours with LNPs carrying ABE8.20-m and B2M sgRNA at different doses. At 72 hours HSCs were either washed or not and plated for expansion until day 7. The intermediate (ID = 1 | .g) and high doses (HD = 1.5 ng) of LNPs reached 70% and 90% efficiency respectively not only in the bulk populations but also in CD90intermediateand CD90hlghHSPCs (Figure 12 B-E). Remarkably, phenotypic composition revealed a toxicity induced by LNPs at the highest dose tested as measured by the disappearance of CD90highHSPCs in absence of the washing step (Figure 12 F). Conversely, a phenotypic composition comparable to untreated cells was preserved when including the washing step in the process. Further to highlighting that LNP delivery of ABE can be integrated in a clinical process, these data further demonstrate that the washing step is crucial to preserve the most primitive compartment endowed with long-term engraftment and hematopoietic reconstitution potential.

Claims

CLAIMS1. An ex vivo or in vitro method for genetic modification of a cell of the immune system or a progenitor thereof, comprising the steps of:- providing a cell of the immune system or a progenitor thereof,- incubating in a culture medium said cell with lipid nanoparticles (LNPs) comprising at least one gene-modifying agent, obtaining a genetically modified cell, wherein said culture medium is serum-free;- removing, after incubation, the LNPs;- optionally collecting the genetically modified cell.

2. The method of claim 1, wherein the genetic modification is gene editing and the at least one gene-modifying agent is a gene editing agent.

3. The method of claim 2, wherein the gene editing agent is a nuclease mRNA or ribonucleoprotein (RNP) comprising a nuclease.

4. The method of claim 3, wherein said nuclease is selected from: zinc-finger nuclease, a transcription activator like effector nuclease (TALENs), and a CRISPR / Cas9 nuclease.

5. The method of claim 2 wherein the gene editing agent is a base editor or a prime editor.

6. The method of claim 1, wherein the genetic modification is epigenetic editing and the at least one gene-modifying agent is an epigenetic editing agent, preferably an artificial transcription repressor (ATR).

7. The method of any one of claims 1-6, wherein the cell is a hematopoietic stem and / or progenitor cell (HSPC).

8. The method of any one of claims 1-6, wherein the cell is a T cell.

9. The method of any one of claims 1-8, further comprising a step of transducing the cells with a viral vector, preferably an adeno-associated vector (AAV), a lentiviral vector, or anintegration-defective lentiviral vector (IDLV), preferably said viral vector comprising an editing template polynucleotide.

10. The method of claim 9, wherein the cell is contacted with the viral vector at the same time as it is contacted with the lipid nanoparticles, or within a time of from 1 minute up to 48 hours after the cell is contacted with the lipid nanoparticles, or 12-48 hours after the cells are contacted with the lipid nanoparticles.

11. The method of claim 9, wherein the cell is transduced with the viral vector within 12 hours, within 8 hours, or within 2 hours, prior to incubation of said cells with said lipid nanoparticles.

12. The method of any one of claims 1-11, wherein the step of incubating the cell with lipid nanoparticles is carried out for an incubation time of from 8 to 48 hours.

13. The method of any one of claims 1-12, wherein the step of incubating the cell with lipid nanoparticles is carried out for an incubation time of from 8 to 30 hours.

14. The method of any one of claims 1-13, wherein the cells are cultured in a serum-free medium at least from two days before incubation and at least for 24 hours during incubation with LNPs.

15. The method of any one of claims 1-13, wherein the cells are cultured in a serum-free medium for 12-48 hours before incubation with the lipid nanoparticles, preferably wherein the cells are cultured in a serum-free medium for 12-48 hours before incubation and for 12-24 hours during incubation with LNPs.

16. The method of any one of claims 1-15, wherein the cells are incubated with a concentration of LNPs of 0.1-5 pg / 500.000 cells, preferably of 0.5-4 pg / 500.000 cells, more preferably of 1- 3 pg / 500.000 cells.

17. The method of any one of claims 1-16, wherein the step of removing, after incubation, the LNPs is carried out by washing the cells with fresh culture medium, preferably with serum-free fresh culture medium.

18. The method of any one of claims 1-17, further comprising culturing the cells in the presence of an inhibitor of senescence, preferably an inhibitor of MAPK / ERK signaling, more preferably a p53 inhibitor19. The method of any one of claims 1-18, wherein the step of removing, after incubation, the LNPs is carried out within 12-48 hours from the starting of the incubation.

20. A population of genetically modified cells obtained by the method of any one of claims 1- 19.

21. A pharmaceutical formulation comprising the population of genetically modified cells of claim 20 and pharmaceutically acceptable carriers, diluents or excipients.

22. The population of genetically modified cells of claim 20 or the pharmaceutical formulation of claim 21, for use as a medicament.

23. The population of genetically modified cells of claim 20 or the pharmaceutical formulation of claim 21, for use in the treatment or prevention of cancer, an immune disorder, a bacterial or viral infection, a genetic disease, a blood disease, a lysosomal storage disease, -thalassemia, Fanconi anemia, bone marrow failure disease, sickle cell disease, osteopetrosis, chronic granulomatous disease, metachromatic leukodystrophy, mucopolysaccharidosis.

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