Gene Therapy

Abstract

23. The use of one or more senescence inhibitors to increase survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

Description

[Technical field]

[0001] The present invention relates to the genetic modification of cells. More specifically, the present invention relates to the use of inhibitors to improve the efficiency of gene editing and to improve the survival and / or engraftment of gene-edited hematopoietic stem cells and / or T cells. [Background technology]

[0002] The hematopoietic system is a complex hierarchy of cells of different mature cell lineages. These include cells of the immune system that provide protection against pathogens, cells that transport oxygen to the body, and cells involved in wound healing. All these mature cells originate from a pool of hematopoietic stem cells (HSCs), which have the capacity to self-renew and differentiate into any blood cell lineage. HSCs have the potential to replenish the entire hematopoietic system.

[0003] Hematopoietic cell transplantation (HCT) is a curative therapy for several genetic and acquired disorders. However, allogeneic HCT is limited by the low availability of matched donors, the mortality associated with the allogeneic procedure, mostly related to graft-versus-host disease (GvHD) and infectious complications caused by severe and long-lasting immunodeficiency.

[0004] Adoptive immunotherapy using genetically engineered cells (e.g., T cells or NK cells) is a promising new clinical strategy. For example, T cells and NK cells genetically modified with transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs) show promise as cancer treatments.

[0005] Gene therapy approaches based on the transplantation of genetically modified autologous HSCs and / or T cells offer potentially improved safety and efficacy over allogeneic HCT and / or allogeneic adoptive immunotherapy, and are particularly relevant for patients without a matched donor.

[0006] The concept of stem cell gene therapy or adoptive immunotherapy is based on the genetic modification of a relatively small number of stem cells, T cells or NK cells. These genetically modified cells undergo self-renewal to persist long-term in the body and generate genetically "corrected" progeny. This ensures a continuous supply of corrected cells for the rest of the patient's life. HSCs are a particularly attractive target for gene therapy because their genetic modifications are passed on to all blood cell lineages as they differentiate. Moreover, HSCs can be easily and safely obtained, for example, from bone marrow, mobilized peripheral blood and umbilical cord blood.

[0007] Efficient and long-term genetic modification of HSCs and their progeny (as well as T cells) requires techniques that allow stable integration of corrective DNA into the genome without affecting HSC function. Thus, the use of integrating recombinant viral systems such as gamma-retroviruses, lentiviruses, and spumaviruses has been central to the field (Chang, AH et al. (2007) Mol. Ther. 15: 445-456). Therapeutic benefits have already been achieved in gamma-retrovirus-based clinical trials for adenosine deaminase-associated severe combined immunodeficiency (ADA-SCID; Aiuti, A. et al. (2009) N. Engl. J. Med. 360: 447-458), X-linked severe combined immunodeficiency (SCID-X1; Hacein-Bey-Abina, S. et al. (2010) N. Engl. J. Med. 363: 355-364) and Wiskott-Aldrich syndrome (WAS; Boztug, K. et al. (2010) N. Engl. J. Med. 363: 1918-1927). Additionally, lentiviruses have been used as delivery vehicles in the treatment of X-linked adrenoleukodystrophy (ALD; Cartier, N. et al. (2009) Science 326: 818-823) and beta-thalassemia (Cartier, N. et al. (2010) Bull. Acad. Natl. Med. 194: 255-264; discussion 264-258), and more recently metachromatic leukodystrophy (MLD; Biffi, A. et al. (2013) Science 341: 1233158) and WAS (Aiuti, A. et al. (2013) Science 341: 1233151).

[0008] In addition to the use of retroviral and lentiviral based vectors, vectors derived from other viruses, such as adenoviruses and adeno-associated viruses (AAV), can also be utilized to modify hematopoietic stem and progenitor cells.

[0009] In recent years, the scope of genetic manipulation has expanded from gene replacement to targeted gene editing using engineered nucleases that allow precise sequence modification of a locus of interest. In other words, hematopoietic stem and / or progenitor cells (HSPCs) and / or T cells can be genetically modified to prevent or treat disease by adding normal genes (gene transfer) or by repairing normal gene defects (gene editing). Applications of gene editing include targeted disruption of gene coding sequences, precise sequence replacement for in situ correction of mutations, and targeted transgene insertion into a given locus. Gene editing is based on the design of artificial endonucleases that target double-strand breaks (DSBs) or nicks to sequences of interest in the genome. Cells repair DSBs through two main mechanisms, non-homologous end joining (NHEJ) or homology-dependent repair (HDR), although other repair mechanisms may eventually be used. While NHEJ often generates small insertions or deletions ("indels") that can disrupt the coding sequence of a gene at the target site, HDR can be used to precisely introduce new sequences at a target site by providing foreign template DNA that has homology to sequences flanking the DSB.

[0010] To target a locus of interest, multiple platforms of artificial endonucleases can be used, including zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and the more recently developed RNA-based CRISPR / Cas9 nucleases. Viral vectors are the most efficient delivery vehicles for DNA templates, and for example, AAV6 vectors can achieve high transduction efficiency in human primary cells, such as HSPCs and T lymphocytes.

[0011] Successful HSPC gene therapy and adoptive immunotherapy critically depend on the ability to genetically modify HSPCs and / or T cells without compromising their functional properties and viability. Current protocols for gene transfer and editing require long-term ex vivo culture, high doses of viral vectors, and nuclease-induced DNA DSBs that activate DNA damage response (DDR) pathways and lead to cell cycle arrest. Emerging data suggest that cellular detection of viral vectors used in classical gene therapy settings unexpectedly triggers DDR rather than eliciting innate immune-mediated recognition of viral nucleic acids or proteins.

[0012] The DDR pathway is an evolutionarily conserved set of actions that converge on key decision-makers, such as the tumor suppressor p53, to enforce cell cycle arrest. Activation of the DDR pathway impairs hematopoietic reconstitution of cells genetically modified by gene addition with lentiviral vectors upon transplantation (Piras, F. et al., 2017, EMBO Mol Med 9: 1198-1211). In line with this, the involvement of the p53 DDR signaling cascade has recently been identified as a barrier to successful gene editing approaches in HSPCs (Schiroli, G. et al., 2019, Cell Stem Cell 24: 551-565; and Conti, A. & Di Micco, R., 2018, Genome Med 10: 66). Even a single nuclease-induced DSB triggers a transient, but detectable, DDR in HSPCs. Unexpectedly, simultaneous exposure to nuclease-induced DSBs and recombinant adeno-associated virus serotype 6 (rAAV6), currently the preferred source for delivering HDR templates during gene editing, led to elevated DDR load and prolonged arrest of HSPC proliferation despite the absence of cell death, resulting in poor engraftment potential of HSPCs after transplantation. Recently, detailed molecular characterization of DDR dynamics combined with single-cell transcriptomic studies led to the development of an innovative strategy based on transient inhibition of p53 during the editing procedure, which improves hematopoietic reconstitution without exacerbating chromosomal translocations or mutation load (Schiroli, G. et al., 2019, Cell Stem Cell 24: 551-565; and WO2020002380). This experimental evidence reveals a previously unknown interplay between viral vector sensing and the host cell DDR machinery and represents the first example of how transient manipulation of the DDR program can affect the biology of gene-corrected HSPCs.

[0013] Substantial challenges remain in the methods employed for genetic modification of hPSCs and T cells. In particular, existing methods require multiple administrations of high doses of vectors and long ex vivo activation times, which may lead to problems in the survival of transduced HSPCs and T cells in culture and affect their biological properties. Furthermore, improved engraftment of transduced cells would be of great benefit for clinical applications. Summary of the Invention

[0014] Summary of the Invention We have been studying the inflammatory response caused by activation of DDR by induction of double-stranded breaks (DSBs) in DNA by nucleases in gene editing / therapeutic engineering technology. We recently found that cellular detection of viral vectors, which are employed in classical gene therapy, unexpectedly induces DDR. Activation of the DDR pathway via induction of DSBs and cellular detection of viral vectors impairs hematopoietic reconstitution of gene-modified cells upon transplantation.

[0015] In addition to the above challenges associated with gene therapy and gene editing approaches in HSPCs and T cells, we hypothesize that current genetic engineering protocols (requiring long activation times in culture, high vector doses, and nuclease-induced DSBs) may inadvertently trigger the activation of a cellular senescence program in HSPCs, with cell-autonomous and paracrine short- and long-term consequences on engineered human hematopoiesis. This program may in turn inhibit the proliferation of genetically modified cells and affect their clonal composition and the kinetics of hematopoietic reconstitution upon transplantation, posing a real challenge to realizing the full potential of gene therapy approaches. The impact of senescence on hematopoietic reconstitution may be further exacerbated when i) the number of ex vivo gene-corrected cells available for transplantation is low, ii) HSPCs are harvested from older donors, and / or iii) the pathophysiology of the underlying disease is intrinsically associated with hematopoietic stress (e.g., Fanconi anemia) or hyperinflammation (e.g., chronic granulomatous diseases).

[0016] The present inventors have developed an improved protocol for culturing hematopoietic stem cells (HSCs), progenitor cells (HSPCs) and T cells engineered with viral vectors (AAV or LV) for gene therapy and / or gene editing. The present inventors have unexpectedly found that the addition of senescence inhibitors, particularly inhibitors targeting the IL-1 and NF-κB signaling pathways, during gene editing and / or transduction with viral vectors attenuates DDR-dependent inflammatory responses and improves the clonogenic potential and long-term reconstitution in vivo of gene-edited and / or transduced cells. The present inventors have also unexpectedly found that pre-incubating cells with senescence inhibitors (which reduce the proportion of senescent cells), particularly p38 MAPK inhibitors, improves the efficacy of gene editing and / or transduction.

[0017] Advantageously, the present invention improves the function of gene edited and / or transduced HSPCs and / or T cells by inhibiting senescence prior to gene editing / transduction and by inhibiting DDR pathways due to DNA DSBs and their associated inflammatory responses during gene editing / transduction.

[0018] Thus, in one aspect, the invention provides the use of one or more senescence inhibitors to increase survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0019] In a further aspect, the present invention provides the use of one or more senescence inhibitors to increase gene editing efficiency of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0020] In a further aspect, the present invention provides one or more senescence inhibitors for use in hematopoietic cell gene therapy, hematopoietic stem cell gene therapy, hematopoietic progenitor cell gene therapy and / or T cell gene therapy.

[0021] In a further aspect, the present invention provides one or more senescence inhibitors for use in increasing survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0022] In a further aspect, the present invention provides the use of one or more senescence inhibitors to preserve or enhance the fitness of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0023] In a further aspect, the present invention provides one or more senescence inhibitors for use in maintaining or enhancing the fitness of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0024] In some embodiments, fitness is maintained or enhanced in gene-edited hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells.

[0025] Preferably, a combination of aging inhibitors is used. Preferably, each of the aging inhibitors in the combination is a different inhibitor. For example, each of the aging inhibitors can target a different molecule, i.e., the inhibitors do not have to target the same molecule.

[0026] In some embodiments, the one or more aging inhibitors are in the form of a composition or kit.

[0027] In some embodiments, the aging inhibitors are in combination. Suitably, the inhibitors may be administered simultaneously, sequentially or separately.

[0028] In some embodiments, the one or more senescence inhibitors comprise or consist of a MAPK / ERK signaling inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor. Suitably, the MAPK / ERK signaling inhibitor is a MAPK inhibitor.

[0029] In some embodiments, the one or more senescence inhibitors comprise or consist of a MAPK inhibitor, an IL-1 inhibitor, and / or an NF-κB inhibitor.

[0030] Thus, in one aspect, the invention provides the use of a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor to increase survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0031] In a further aspect, the present invention provides the use of a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor to increase gene editing efficiency of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0032] In a further aspect, the present invention provides a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor for use in hematopoietic cell gene therapy, hematopoietic stem cell gene therapy, hematopoietic progenitor cell gene therapy and / or T cell gene therapy.

[0033] In a further aspect, the present invention provides a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor for use in the survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0034] In some embodiments, gene therapy comprises gene transfer.

[0035] In some embodiments, gene therapy comprises gene editing.

[0036] In some embodiments, the one or more aging inhibitors comprise or consist of a MAPK inhibitor. Preferably, a MAPK inhibitor is used.

[0037] In some embodiments, the one or more senescence inhibitors comprise or consist of an IL-1 inhibitor. Preferably, an IL-1 inhibitor is used.

[0038] In some embodiments, the one or more senescence inhibitors comprise or consist of an NF-κB inhibitor. Preferably, an NF-κB inhibitor is used.

[0039] In some embodiments, the one or more senescence inhibitors comprise or consist of a MAPK inhibitor and an IL-1 inhibitor. Preferably, a MAPK inhibitor and an IL-1 inhibitor are used.

[0040] In some embodiments, the one or more senescence inhibitors comprise or consist of a MAPK inhibitor and an NF-κB inhibitor. Preferably, a MAPK inhibitor and an NF-κB inhibitor are used.

[0041] In some embodiments, the one or more senescence inhibitors comprise or consist of an IL-1 inhibitor and an NF-κB inhibitor. Preferably, an IL-1 inhibitor and an NF-κB inhibitor are used.

[0042] In some embodiments, the one or more senescence inhibitors comprise or consist of a MAPK inhibitor, an IL-1 inhibitor, and an NF-κB inhibitor. Preferably, a MAPK inhibitor, an IL-1 inhibitor, and an NF-κB inhibitor are used.

[0043] In one embodiment, the MAPK / ERK signaling inhibitor (eg, a MAPK inhibitor), an IL-1 inhibitor and / or an NF-κB inhibitor are administered simultaneously, sequentially or separately.

[0044] In some embodiments, the IL-1 inhibitor and / or NF-κB inhibitor inhibits DDR-dependent inflammation. Suitably, the inhibition of DDR-dependent inflammation increases the survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells in gene therapy. Suitably, the inhibition of DDR-dependent inflammation increases gene editing efficiency of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0045] In one embodiment, when cells are exposed to the inhibitor, cultured cell viability is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 50%, 75% or 90%, preferably at least 70%, greater than in its absence for a period of time (e.g., about 6 or 12 hours, or 1, 2, 3, 4, 5, 6, 7 or more days, preferably about 2 days).

[0046] The present invention allows for a reduction in the timing of cell pretreatment / conditioning with cytokines to prime (otherwise quiescent) cells for transduction. Standard timing for HSPCs is 3 days. This time can be reduced to less than 3 days, or less than 2 days, or less than 1 day, e.g., 0, 1 or 2 days.

[0047] In one embodiment, engraftment of transplanted hematopoietic stem and / or hematopoietic progenitor cells and / or their progeny (e.g., graft-derived cells) in a host subject is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or 75%, preferably at least 10%, greater when the cells are exposed to an inhibitor than in its absence.

[0048] In one embodiment, the cells are HSCs.

[0049] In one embodiment, the cells are HSPCs.

[0050] In one embodiment, the HSPCs are CD34 + It is a cell.

[0051] In one embodiment, the population of hematopoietic stem and / or progenitor cells is CD34 + Contains cells or CD34 + Cells are enriched or CD34 + The cell population consists essentially of specific subpopulations of cells, e.g., CD34 + CD38 -The cell population may be further enriched for a particular subpopulation of cells, e.g., CD34 + CD133 + and CD90 + It may be further enriched for cells.

[0052] In some embodiments, the MAPK / ERK signaling inhibitor is a MAP3K inhibitor, a MAK2K inhibitor, a MAPK inhibitor, preferably an MKK7 inhibitor, an MKK4 inhibitor, an MKK3 / 6 inhibitor, a MEK1 / 2 inhibitor, a JNK inhibitor, a p38 inhibitor or an ERK inhibitor.

[0053] In some embodiments, the MAPK inhibitor is a p38 phosphorylation inhibitor, a JNK phosphorylation inhibitor or an ERK phosphorylation inhibitor, preferably a p38 phosphorylation inhibitor.

[0054] In some embodiments, the MAPK inhibitor is a JNK inhibitor, a p38 inhibitor, or an ERK inhibitor.

[0055] In some embodiments, the MAPK inhibitor is FR180204, SP600125, SB203580, SB202190, LY2228820, BIRB796; SB203580 hydrochloride, SCIO 469 hydrochloride, TMCB, XMD8-92, TCS JNK 6o, SU3327, CC401 dihydrochloride, or a derivative thereof.

[0056] In some embodiments, the MAPK inhibitor is FR180204, SP600125, SB203580, or a derivative thereof.

[0057] In some embodiments, the IL-1 inhibitor is an anti-IL-1α antibody, an anti-IL-1β antibody, an IL-1 antagonist, an IL-1 receptor antagonist, an IL-1α converting enzyme inhibitor, an IL-1β converting enzyme inhibitor, or a soluble decoy IL-1 receptor.

[0058] In some embodiments, the IL-1 inhibitor is anakinra, canakinumab, rilonacept, gevokizumab, or variants thereof.

[0059] In some embodiments, the IL-1 inhibitor is anakinra or a variant thereof.

[0060] In some embodiments, the NF-κB inhibitor is an IL-1 inhibitor, an IL-1 receptor inhibitor, a TLR4 inhibitor, a TAK1 inhibitor, an Akt inhibitor, an IKK inhibitor, an IκB phosphorylation inhibitor, an IκB degradation inhibitor, a proteasome inhibitor, an IκBα upregulation inhibitor, an NF-κB nuclear translocation inhibitor, an NF-κB expression inhibitor, an NF-κB DNA binding inhibitor, or an NF-κB transactivation inhibitor.

[0061] In some embodiments, the NF-κB inhibitor is SC514 or a derivative thereof; anakinra, canakinumab, rilonacept, gevokizumab or a variant thereof; or metformin, apigenin, kaempferol, BAY11-7082, or a derivative thereof.

[0062] In some embodiments, the NF-κB inhibitor is SC514 or a derivative thereof.

[0063] In some embodiments, the use further comprises the use of an agent that promotes homology dependent DNA repair.

[0064] In some embodiments, the agent is a p53 activation inhibitor, preferably, the inhibitor is a p53 phosphorylation inhibitor, more preferably, a p53 serine 15 phosphorylation inhibitor.

[0065] In some embodiments, the p53 activation inhibitor is a p53 dominant negative peptide, an ataxia telangiectasia mutated (ATM) kinase inhibitor or an ataxia telangiectasia and Rad3-related protein (ATR) inhibitor.

[0066] In some embodiments, the p53 activation inhibitor is pifithrin-α or a derivative thereof; KU-55933 or a derivative thereof; GSE56 or a mutant thereof; KU-60019, BEZ235, wortmannin, CP-466722, torin2, CGK733, KU-559403, AZD6738 or a derivative thereof; or siRNA, shRNA, miRNA or antisense DNA / RNA, preferably, the p53 activation inhibitor is GSE56 or a mutant thereof.

[0067] In some embodiments, the inhibition of senescence in hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (eg, inhibition of MAPK, inhibition of IL-1 and / or inhibition of NF-κB) is transient.

[0068] In one embodiment, the one or more senescence inhibitors are transient inhibitors, such as reversible inhibitors (e.g., having an inhibitory effect lasting less than about 1, 2, 3, 4, 5, 6, 7, or 14 days). Preferably, the cells are exposed to the inhibitor for about 1-48 or 1-24 hours, preferably 1-24 hours. The cells may be exposed to the inhibitor before, simultaneously with, or after, for example, the viral vector and / or gene editing mechanism.

[0069] In some embodiments, the inhibition of p53 activation in hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells is transient. In one embodiment, the p53 activation inhibitor is a transient inhibitor, such as a reversible inhibitor (e.g., having an inhibitory effect lasting about 1, 2, 3, 4, 5, 6, 7, or 14 days). Preferably, the cells are exposed to the inhibitor for about 1 to 48 or 1 to 24 hours, preferably 1 to 24 hours. The cells may be exposed to the inhibitor, for example, before, simultaneously with, or after the viral vector and / or gene editing mechanism.

[0070] In some embodiments, the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are exposed to an IL-1 inhibitor and / or an NF-κB inhibitor before, concomitantly with and / or after the gene editing mechanism is introduced into the cells, preferably simultaneously with the gene editing mechanism being introduced into the cells.

[0071] In some embodiments, inhibition of IL-1 and / or NF-κB occurs during gene editing of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells.

[0072] In some embodiments, hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are exposed to a MAPK inhibitor before the gene editing machinery is introduced into the cells.

[0073] In some embodiments, MAPK inhibition occurs prior to and / or during gene editing of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0074] In some embodiments, hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are exposed to a p53 activation inhibitor before, concomitantly with and / or after the gene editing mechanism is introduced into the cells.

[0075] In some embodiments, inhibition of p53 occurs during gene editing of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells.

[0076] In some embodiments, one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor, and / or an NF-κB inhibitor) are added to the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells at a concentration of about 0.5-200 μM or about 0.1-200 ng / μL.

[0077] In one embodiment, one or more senescence inhibitors (e.g., MAPK inhibitors, IL-1 inhibitors and / or NF-κB inhibitors) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 0.5 to 200, 0.5 to 150, 0.5 to 100, 0.5 to 50, 0.5 to 40, 0.5 to 30, 0.5 to 20, or 0.5 to 15 μM, preferably about 0.5 to 30 μM, more preferably about 0.5 to 15 μM. In one embodiment, one or more senescence inhibitors (e.g., MAPK inhibitors, IL-1 inhibitors, and / or NF-κB inhibitors) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 1-200, 1-150, 1-100, 1-50, 1-40, 1-30, 1-20, or 1-15 μM, preferably about 1-30 μM, more preferably about 1-15 μM. In another embodiment, the inhibitors are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells at a concentration of about 5-200, 5-150, 5-100, 5-50, 5-40, 5-30, 5-20, or 5-15 μM, preferably about 5-30 μM.

[0078] In one embodiment, one or more senescence inhibitors (e.g., MAPK inhibitors, IL-1 inhibitors, and / or NF-κB inhibitors) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, or 200 μM. In another embodiment, one or more senescence inhibitors (e.g., MAPK inhibitors, IL-1 inhibitors, and / or NF-κB inhibitors) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 2 μM. In another embodiment, one or more senescence inhibitors (e.g., MAPK inhibitors, IL-1 inhibitors, and / or NF-κB inhibitors) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 4 μM. In another embodiment, one or more senescence inhibitors are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 12 μM.

[0079] In one embodiment, one or more senescence inhibitors (e.g., MAPK inhibitors, IL-1 inhibitors and / or NF-κB inhibitors) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 0.1 to 200, 0.1 to 150, 0.1 to 100, 0.1 to 75, 0.1 to 60, 0.1 to 50, 0.1 to 25, 0.1 to 20, 0.1 to 15, or 0.1 to 10 ng / μL, preferably about 0.1 to 60 ng / μL. In another embodiment, the inhibitor is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells at a concentration of about 5 to 200, 5 to 150, 5 to 100, 5 to 75, 5 to 60, 5 to 50, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 ng / μL, preferably about 5 to 60 ng / μL.

[0080] In one embodiment, one or more senescence inhibitors (e.g., MAPK inhibitors, IL-1 inhibitors and / or NF-κB inhibitors) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, or 200 ng / μL, preferably about 50 ng / μL.

[0081] In some embodiments, one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor, and / or an NF-κB inhibitor) are used in combination with at least one adenoviral protein or a nucleic acid sequence encoding same.

[0082] In some embodiments, the one or more senescence inhibitors (eg, a MAPK inhibitor, an IL-1 inhibitor, and / or an NF-κB inhibitor) further comprise at least one adenoviral protein.

[0083] In some embodiments, the one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor, and / or an NF-κB inhibitor) further comprise a nucleic acid sequence encoding at least one adenoviral protein.

[0084] In some embodiments, the adenoviral proteins are transiently expressed in hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells, preferably, the transient expression occurs during gene editing of the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells.

[0085] In some embodiments, the gene editing target is selected from the group consisting of FANC-A, CD40L, RAG-1, IL-2RG, CYBA, CYBB, NCF1, NCF2, and NCF4. In one embodiment, the gene editing target is a gene mutated in chronic granulomatous disease, or a gene mutated in genetically mutated SCID, atypical SCID and Omenn's syndrome, or Hyper IgM syndrome.

[0086] In a further aspect, the invention provides a method for gene editing of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells, comprising: (a) contacting a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells with one or more senescence inhibitors; (b) introducing gene editing machinery into a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells using one or more vectors; and (c) editing the genome of said hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells. The present invention provides a method comprising:

[0087] In a further aspect, the invention provides a method for gene editing of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells, comprising: (a) contacting a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor; (b) introducing gene editing machinery into a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells using one or more vectors; and (c) editing the genome of said hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells. The present invention provides a method comprising:

[0088] In a further aspect, the invention provides a method for transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with a viral vector, comprising: (a) contacting a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more senescence inhibitors; and (b) transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more viral vectors. The present invention provides a method comprising:

[0089] In a further aspect, the invention provides a method for transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with a viral vector, comprising: (a) contacting a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells with a MAPK inhibitor, an IL-1 inhibitor, and / or an NF-κB inhibitor; and (b) transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more viral vectors. The present invention provides a method comprising:

[0090] In some embodiments, the method increases the transduction efficiency of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells. An increase in transduction efficiency can be, for example, an increase in vector copy number per cell (e.g., at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 3-fold or more increase). An increase in transduction efficiency can be, for example, an increase in the percentage of cells that are transduced (e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more increase).

[0091] In some embodiments, the steps of introducing a gene editing mechanism into the cell population and contacting the cell population with one or more senescence inhibitors are performed ex vivo or in vitro.

[0092] In some embodiments, the steps of transducing the cell population and contacting the cell population with one or more senescence inhibitors are performed ex vivo or in vitro.

[0093] In one embodiment, the cells are HSCs.

[0094] In one embodiment, the cells are HSPCs.

[0095] In one embodiment, HSPCs are CD34 + It is a cell.

[0096] In one embodiment, the population of hematopoietic stem and / or progenitor cells is CD34 + Contains cells or CD34 + Cells are enriched or CD34 + The cell population consists essentially of specific subpopulations of cells, e.g., CD34 + CD38 - The cell population may be further enriched for a particular subpopulation of cells, e.g., CD34 + CD133 + and CD90 + It may be further enriched for cells.

[0097] In some embodiments, the one or more senescence inhibitors comprise or consist of a MAPK inhibitor, an IL-1 inhibitor, and / or an NF-κB inhibitor.

[0098] In some embodiments, the one or more senescence inhibitors comprise or consist of a MAPK inhibitor.

[0099] In some embodiments, the one or more senescence inhibitors comprise or consist of an IL-1 inhibitor.

[0100] In some embodiments, the one or more senescence inhibitors comprise or consist of an NF-κB inhibitor.

[0101] In some embodiments, the one or more senescence inhibitors comprise or consist of a MAPK inhibitor and an IL-1 inhibitor.

[0102] In some embodiments, the one or more senescence inhibitors comprise or consist of a MAPK inhibitor and a NF-κB inhibitor.

[0103] In some embodiments, the one or more senescence inhibitors comprise or consist of an IL-1 inhibitor and an NF-κB inhibitor.

[0104] In some embodiments, the one or more senescence inhibitors comprise or consist of a MAPK inhibitor, an IL-1 inhibitor, and an NF-κB inhibitor.

[0105] In some embodiments, the MAPK / ERK signaling inhibitor is a MAP3K inhibitor, a MAK2K inhibitor, a MAPK inhibitor, preferably an MKK7 inhibitor, an MKK4 inhibitor, an MKK3 / 6 inhibitor, a MEK1 / 2 inhibitor, a JNK inhibitor, a p38 inhibitor or an ERK inhibitor.

[0106] In some embodiments, the MAPK inhibitor is a p38 phosphorylation inhibitor, a JNK phosphorylation inhibitor or an ERK phosphorylation inhibitor, preferably a p38 phosphorylation inhibitor.

[0107] In some embodiments, the MAPK inhibitor is a JNK inhibitor, a p38 inhibitor, or an ERK inhibitor.

[0108] In some embodiments, the MAPK inhibitor is FR180204, SP600125, SB203580, SB202190, LY2228820, BIRB796; SB203580 hydrochloride, SCIO 469 hydrochloride, TMCB, XMD8-92, TCS JNK 6o, SU3327, CC401 dihydrochloride, or a derivative thereof.

[0109] In some embodiments, the MAPK inhibitor is FR180204, SP600125, SB203580, or a derivative thereof.

[0110] In some embodiments, the IL-1 inhibitor is an anti-IL-1α antibody, an anti-IL-1β antibody, an IL-1 antagonist, an IL-1 receptor antagonist, an IL-1α converting enzyme inhibitor, an IL-1β converting enzyme inhibitor, or a soluble decoy IL-1 receptor.

[0111] In some embodiments, the IL-1 inhibitor is anakinra, canakinumab, rilonacept, gevokizumab, or a variant thereof.

[0112] In some embodiments, the IL-1 inhibitor is anakinra or a variant thereof.

[0113] In some embodiments, the NF-κB inhibitor is an IL-1 inhibitor, an IL-1 receptor inhibitor, a TLR4 inhibitor, a TAK1 inhibitor, an Akt inhibitor, an IKK inhibitor, an IκB phosphorylation inhibitor, an IκB degradation inhibitor, a proteasome inhibitor, an IκBα upregulation inhibitor, an NF-κB nuclear translocation inhibitor, an NF-κB expression inhibitor, an NF-κB DNA binding inhibitor, or an NF-κB transactivation inhibitor.

[0114] In some embodiments, the NF-κB inhibitor is SC514 or a derivative thereof; anakinra, canakinumab, rilonacept, gevokizumab or a variant thereof; or metformin, apigenin, kaempferol, BAY11-7082, or a derivative thereof.

[0115] In some embodiments, the NF-κB inhibitor is SC514 or a derivative thereof.

[0116] In one embodiment, the MAPK / ERK signaling inhibitor (eg, a MAPK inhibitor), an IL-1 inhibitor and / or an NF-κB inhibitor are administered simultaneously, sequentially or separately.

[0117] In some embodiments, the population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells is contacted with a MAPK / ERK signaling inhibitor (e.g., a MAPK inhibitor) prior to or simultaneously with the step of introducing a gene editing mechanism into the cells, preferably prior to the step of introducing a gene editing mechanism into the cells.

[0118] In some embodiments, a population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells is contacted with an IL-1 inhibitor and / or an NF-κB inhibitor prior to, simultaneously with, or after introducing a gene editing mechanism into the cells.

[0119] In some embodiments, the population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells is contacted with a MAPK / ERK signaling inhibitor (e.g., a MAPK inhibitor) prior to or simultaneously with the step of transducing the cells, preferably prior to the step of transducing the cells.

[0120] In some embodiments, a population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells is contacted with an IL-1 inhibitor and / or an NF-κB inhibitor prior to, simultaneously with, or after transducing the cells.

[0121] Thus, in one embodiment of the method of gene editing or transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells, steps (a) and (b) can be performed simultaneously, while in another embodiment, steps (a) and (b) can be performed sequentially, either with step (a) being performed before step (b), or with step (b) being performed before step (a).

[0122] In one embodiment, the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are contacted with one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor, preferably a MAPK inhibitor) for about 15 minutes to about 72 hours; about 15 minutes to about 48 hours; or about 15 minutes to about 24 hours; about 15 minutes to about 4 hours; about 15 minutes to about 3 hours; about 15 minutes to about 2 hours; about 15 minutes to about 1 hour prior to transducing the cell population with the viral vector and / or prior to introducing the gene editing mechanism into the cells. In another embodiment, the cells are contacted with the inhibitor for about 1 hour to about 72 hours; about 1 hour to about 48 hours; or about 1 hour to about 24 hours prior to transducing the cell population with the viral vector and / or prior to introducing the gene editing mechanism into the cells. In another embodiment, the cells are contacted with the inhibitor for about 1-4 hours; 1-3 hours; or 1-2 hours prior to transducing the cell population with the viral vector and / or prior to introducing the gene editing mechanism into the cells.

[0123] In one embodiment, hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are contacted with one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor, preferably a MAPK inhibitor) for about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours, preferably about 24 hours or 48 hours, prior to transducing the cell population with a viral vector and / or prior to introducing a gene editing mechanism into the cells.

[0124] In one embodiment, hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are contacted with one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor, preferably a MAPK inhibitor) for about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours or 4 hours, preferably about 15 minutes, prior to transducing the cell population with a viral vector and / or prior to introducing a gene editing mechanism into the cells.

[0125] In one embodiment, the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are contacted with one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor, preferably an IL-1 inhibitor and / or an NF-κB inhibitor) for about 15 minutes to about 4 hours; about 15 minutes to about 3 hours; or about 15 minutes to about 2 hours after transducing the cell population with one or more viral vectors and / or introducing a gene editing mechanism into the cells. In another embodiment, the cells are contacted with the inhibitors for about 1 to 4 hours; 1 to 3 hours; or 1 to 2 hours after transducing the cell population with the viral vectors and / or introducing a gene editing mechanism into the cells.

[0126] In one embodiment, the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are contacted with one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor, preferably an IL-1 inhibitor and / or an NF-κB inhibitor) for about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours or 4 hours, preferably about 15 minutes, after transduction of the cell population with a viral vector and / or after introduction of a gene editing mechanism into the cells (e.g., after electroporation of the cells).

[0127] Suitably, the inhibitor may be active during gene editing.

[0128] Suitably, the inhibitor may be active during transduction.

[0129] In one embodiment, the contacting step is performed about 12 to 60 hours, e.g., 24 to 60 hours, 36 to 60 hours, or 42 to 54 hours, preferably about 42 to 54 hours, before the step of introducing a gene editing mechanism and / or transducing with a viral vector. In one embodiment, the contacting step is performed about 12, 18, 24, 30, 36, 42, 48, 54, or 60 hours, preferably about 48 hours, before the introduction step is initiated.

[0130] In one embodiment, the contacting step is performed for about 12 to 96 hours, e.g., 12 to 60 hours, 24 to 60 hours, 36 to 60 hours, or 42 to 54 hours, preferably about 42 to 54 hours, after the step of introducing the gene editing mechanism and / or transducing with the viral vector. In one embodiment, the contacting step is performed for about 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 hours, preferably about 48 hours or about 96 hours, after the introducing and / or transducing step.

[0131] In one embodiment, the contacting step is performed for about 12 to 96 hours, e.g., 12 to 72 hours, 12 to 60 hours, 24 to 60 hours, 36 to 60 hours, or 42 to 54 hours, preferably about 72 to 96 hours, after initiating culture of the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., initiating culture after the cells are thawed from a frozen state). In one embodiment, the contacting step is performed for about 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 hours, preferably about 72 hours, after initiating culture of the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0132] In one embodiment, the contacting step is performed for about 12 to 96 hours, for example, 12 to 60 hours, 24 to 60 hours, 36 to 60 hours, or 42 to 54 hours, preferably about 72 to 96 hours, after thawing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., stored in a frozen state). In one embodiment, the contacting step is performed for about 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, or 102 hours, preferably about 72 hours, after thawing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0133] In some embodiments, the method further comprises contacting the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with an agent that promotes homology-dependent DNA repair, preferably, the agent is a p53 activation inhibitor.

[0134] In some embodiments, the agent is a p53 activation inhibitor, preferably the inhibitor is a p53 phosphorylation inhibitor, more preferably a p53 serine 15 phosphorylation inhibitor.

[0135] In some embodiments, the p53 activation inhibitor is a p53 dominant negative peptide, an ataxia telangiectasia mutated (ATM) kinase inhibitor or an ataxia telangiectasia and Rad3-related protein (ATR) inhibitor.

[0136] In some embodiments, the p53 activation inhibitor is pifithrin-α or a derivative thereof; KU-55933 or a derivative thereof; GSE56 or a mutant thereof; KU-60019, BEZ235, wortmannin, CP-466722, torin2, CGK733, KU-559403, AZD6738 or a derivative thereof; or siRNA, shRNA, miRNA or antisense DNA / RNA, preferably, the p53 activation inhibitor is GSE56 or a mutant thereof.

[0137] In some embodiments, one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor, and / or an NF-κB inhibitor) are added to the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells at a concentration of about 0.5-200 μM or about 0.1-200 ng / μL.

[0138] In one embodiment, one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor, preferably an IL-1 inhibitor and / or an NF-κB inhibitor) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 0.5 to 200, 0.5 to 150, 0.5 to 100, 0.5 to 50, 0.5 to 40, 0.5 to 30, 0.5 to 20, or 0.5 to 15 μM, preferably about 0.5 to 30 μM, more preferably about 0.5 to 15 μM. In one embodiment, one or more senescence inhibitors (e.g., MAPK inhibitors, IL-1 inhibitors and / or NF-κB inhibitors, preferably IL-1 inhibitors and / or NF-κB inhibitors) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 1-200, 1-150, 1-100, 1-50, 1-40, 1-30, 1-20 or 1-15 μM, preferably about 1-30 μM, more preferably about 1-15 μM. In another embodiment, the inhibitors are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells at a concentration of about 5-200, 5-150, 5-100, 5-50, 5-40, 5-30, 5-20 or 5-15 μM, preferably about 5-30 μM.

[0139] In one embodiment, one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor, preferably an IL-1 inhibitor and / or an NF-κB inhibitor) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175 or 200 μM. In another embodiment, one or more senescence inhibitors (e.g., MAPK inhibitors, IL-1 inhibitors and / or NF-κB inhibitors, preferably IL-1 inhibitors and / or NF-κB inhibitors) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 2 μM. In another embodiment, one or more senescence inhibitors (e.g., MAPK inhibitors, IL-1 inhibitors and / or NF-κB inhibitors, preferably IL-1 inhibitors and / or NF-κB inhibitors) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 4 μM. In another embodiment, one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor, preferably an IL-1 inhibitor and / or an NF-κB inhibitor) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture).

[0140] In one embodiment, one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor, preferably an IL-1 inhibitor and / or an NF-κB inhibitor) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 0.1 to 200, 0.1 to 150, 0.1 to 100, 0.1 to 75, 0.1 to 60, 0.1 to 50, 0.1 to 25, 0.1 to 20, 0.1 to 15, or 0.1 to 10 ng / μL, preferably about 0.1 to 60 ng / μL. In another embodiment, the inhibitor is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cell cultures at a concentration of about 5-200, 5-150, 5-100, 5-75, 5-60, 5-50, 5-25, 5-20, 5-15, or 5-10 ng / μL, preferably about 5-60 ng / μL.

[0141] In one embodiment, one or more senescence inhibitors (e.g., a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor, preferably an IL-1 inhibitor and / or an NF-κB inhibitor) are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, or 200 ng / μL, preferably about 50 ng / μL.

[0142] In some embodiments, the method further comprises contacting the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with at least one adenoviral protein or a nucleic acid sequence encoding same.

[0143] In some embodiments, the inhibitor further comprises at least one adenoviral protein.

[0144] In some embodiments, the inhibitor further comprises a nucleic acid sequence encoding at least one adenoviral protein.

[0145] In some embodiments, the adenoviral proteins are transiently expressed in hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells, preferably, the transient expression occurs during gene editing and / or transduction of the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells.

[0146] In some embodiments, the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are obtained from mobilized peripheral blood, bone marrow or umbilical cord blood.

[0147] In some embodiments, the method comprises the further step of enriching the population for hematopoietic stem and / or progenitor cells and / or T cells.

[0148] In some embodiments, the gene editing target is selected from the group consisting of FANC-A, CD40L, RAG-1, IL-2RG, CYBA, CYBB, NCF1, NCF2, and NCF4. In one embodiment, the gene editing target is a gene mutated in chronic granulomatous disease or a gene mutated SCID, atypical SCID and Omenn's syndrome, or Hyper IgM syndrome.

[0149] In a further aspect, the present invention provides a method for producing a method for the treatment of a cancer (a) gene editing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells according to the methods of the invention; and (b) administering to the subject gene-edited hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells. The present invention provides a method of gene therapy comprising:

[0150] In some embodiments, the gene-edited cells are administered to a subject as part of an autologous or allogeneic stem cell transplant procedure.

[0151] In some embodiments, the methods increase gene editing efficiency of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells.

[0152] In some embodiments, the methods increase survival and / or engraftment of populations of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells.

[0153] In some embodiments, the methods increase gene therapy efficiency.

[0154] In a further aspect, the present invention provides a method for producing a method for the treatment of a cancer (a) transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells according to the methods of the invention; and (b) administering to a subject the population of transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells. The present invention provides a method of gene therapy comprising:

[0155] In some embodiments, step (b) comprises administering the transduced cells to the subject as part of an autologous or allogeneic stem cell transplant procedure.

[0156] In some embodiments, the methods increase survival and / or engraftment of populations of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells.

[0157] In some embodiments, the method increases gene therapy efficiency. In some embodiments, the method increases the transduction efficiency of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells. An increase in transduction efficiency can be, for example, an increase in vector copy number per cell (e.g., at least a 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 3-fold or more increase). An increase in transduction efficiency can be, for example, an increase in the percentage of transduced cells (e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more increase).

[0158] The method of gene therapy may be, for example, a method of treating a disease selected from the group consisting of mucopolysaccharidosis type I (MPS-1), chronic granulomatous disease, Fanconi anemia (FA), sickle cell disease, metachromatic leukodystrophy (MLD), globoid cell leukodystrophy (GLD), GM2 gangliosidosis, thalassemia and cancer.

[0159] The method of gene therapy may be, for example, a method of treating diseases caused by Rag-1 mutations, such as SCID, atypical SCID and Omenn's syndrome.

[0160] In one embodiment, the subject is a mammalian subject, preferably a human subject.

[0161] In a further aspect, the present invention provides a population of gene edited and / or transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells prepared according to the methods of the present invention.

[0162] In a further aspect, the present invention provides a pharmaceutical composition comprising a population of gene edited and / or transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells of the present invention.

[0163] In a further aspect, the present invention provides a population of gene edited and / or transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells of the present invention for use in therapy.

[0164] In some embodiments, the population is administered as part of an autologous or allogeneic stem cell transplant procedure. [Brief description of the drawings]

[0165] [Figure 1]Figure 1. A) Schematic of the experimental design. B) Percentage of HDR-edited alleles by digital droplet PCR (ddPCR) (HS / AAV6: n=20; HS / AAV6+ANAK: n=15). ns, P>0.05, Mann-Whitney test. C) Percentage of GFP+ cells within HSPC subpopulations, from more undifferentiated (CD90+) to more differentiated (CD133-). n=8-9; ns, P>0.05, Mann-Whitney test. D) Relative expression of CDKN1A (p21) in HS / AAV6-edited HSPCs 24 and 96 hours after electroporation (n=3, 2, 4, 5). ns, P>0.05, Mann-Whitney test. E) Number of colonies formed by HSPCs treated with -DSB and HS / AAV6 in the presence or absence of anakinra, 24 and 96 hours after electroporation. Kruskal-Wallis test. F) Culture composition of different subpopulations of edited HSPCs 24 and 96 hours after gene editing. [Diagram 2] Figure 2. A) GSEA of whole transcriptome analysis 24 and 96 hours after gene editing in the presence or absence of anakinra. B) GSEA plot of two of the most upregulated inflammatory pathways from the Hallmark category. C) Relative expression of IL1A, IL8, and IL6 in HS / AAV6-edited HSPCs 24 and 96 hours after electroporation (n=9, 2, 9, 3). Mann-Whitney test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Diagram 3]Figure 3. A) Percentage of human CD45+ cells in peripheral blood (PB) of NSG mice transplanted with 3x105 HSPCs electroporated with HS RNP+AAV6 in the presence or absence of anakinra (ANAK). Mean+SEM. ns, P>0.05, Linear Mixed Model (LME) at 18 weeks. B) Percentage of human edited cells in peripheral blood (PB) of NSG mice transplanted as shown in A). *p<0.05; **p<0.01, Linear Mixed Model (LME) at 18 weeks. C) Percentage of human CD45+ cells in bone marrow (BM) of NSG mice transplanted with 3x105 HSPCs electroporated with HS RNP+AAV6 in the presence or absence of anakinra (ANAK). Mean+SEM. ns, P>0.05, Linear Mixed Model (LME) at 18 weeks. ns, P>0.05, Kruskal-Wallis test. D) Percentage of human edited cells in the bone marrow (BM) of NSG mice engrafted as indicated in C). **p<0.01, LME at 18 weeks. E) Percentage of edited subpopulations (HSPC, myeloid cells and B cells) in the bone marrow (BM) of NSG mice engrafted as indicated in C). **p<0.01, LME at 18 weeks. F) Number of colonies formed by BM-derived CD34+ cells treated as in C). *p<0.05; **p<0.01, Kruskal-Wallis test. G) Relative expression of IL8 and CXCL10 in HS / AAV6 BM-derived edited HSPCs with the indicated treatments. ns, P>0.05, *p<0.05, Mann-Whitney test. H) Number of dominant unique BARs in human BM-derived cells at the end of the experiment (18 weeks). **p<0.01; ***p<0.001, Kruskal-Wallis test. I) Number of dominant unique BARs in human PB cells 8-9, 12 and 15 weeks after transplantation into NSG mice. *p<0.05, Mann-Whitney test. [Figure 4] Figure 4. A) Quantification of immunofluorescence staining of nuclear NF-κB (>100 nuclei analyzed) 24 and 96 hours after gene editing under the indicated conditions. B) Representative images of the quantification in A). [Diagram 5]Figure 5. A) Schematic of the experimental design. B) Percentage of HDR edited alleles by digital droplet PCR (ddPCR) (n=20, 11, 3, 5). ns, P>0.05, *P<0.05, Mann-Whitney test. C) Relative expression of IL8, IL6 and CCL2 upon editing in the presence or absence of SC-514 or a combination of GSE56 and SC-514. D) Number of colonies formed by treated HSPCs 96 hours after electroporation. *P<0.05, Kruskal-Wallis test. E) Percentage of human CD45+ cells in peripheral blood (PB) of NSG mice transplanted with 1.5x105 HSPCs electroporated with HS RNP+AAV6 in the presence of GSE56, SC-514 or GSE56+SC-514. Mean+SEM. **p<0.01; ****p<0.0001. Linear mixed model (LME) at 15 weeks. F) Percentage of human CD45+ cells in bone marrow (BM) of NSG mice transplanted with 1,5x105 HSPCs electroporated with HS RNP+AAV6 in the presence of GSE56, SC-514 or GSE56+SC-514. Mean+SEM. *P<0.05, **p<0.01, Kruskal-Wallis test. G) Number of dominant unique BARs in human BM-derived cells at the end of the experiment (15 weeks). **p<0.01, Kruskal-Wallis test. H) Number of dominant unique BARs in human PB cells at 8-9, 12 and 15 weeks after transplantation in NSG mice. **p<0.01, Kruskal-Wallis test. [Figure 6] Figure 6. A) Schematic of the experimental design. B) Number of colonies formed by HSPCs treated with DMSO, 4 μM or 8 μM p38i at 24 h after electroporation. (+DSB(HS): n=7, 7, 3; HS / AAV6: n=10, 10, 5). Kruskal-Wallis test. C) Quantification of cytosolic ROS detected by CM-H2DCFDA and D) mitochondrial superoxide by MitoSOX in HS / AAV6-edited HSPCs at 24 and 96 h after electroporation (C: n=3, 4, 3, 3; D: n=2). Mann-Whitney test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Figure 7] Figure 7. A) Schematic of the experimental design. B) Percentage of human CD45+ cells in peripheral blood (PB) and C) Percentage of human CD45+ cells in bone marrow (BM) of NSG mice transplanted with 1–1.5 × 105 HSPCs pretreated with DMSO or 4 μM p38i and then electroporated with HS RNP or HS RNP+AAV6. Mean + SEM. D) Number of dominant unique BARs in human BM-derived cells at the end of the experiment (week 15). Median. (n=4, 5). E) Schematic of the experimental design. F) Number of colonies formed by CD34+ purified from BM at week 15. HSPCs were treated with DMSO or 4 μM p38i before electroporation (n=7, 10, 9, 8 in two independent experiments). G) Percentage of fluorescent β-galactosidase positive cells measured in CD34+ cells purified from BM at week 15. Mann-Whitney test, *P<0.05; ***P<0.001; ****P<0.0001. [Figure 8] Figure 8. A) Schematic of the experimental design. B) GFP expression 96 hours after editing in different HSPC subpopulations (CD34+CD133-; CD34+CD133+ and CD34+CD133+CD90+ cells) edited upon different treatments (DMSO; 4 μM ERKi; 2 μM JNKi). C) Number of colonies formed 24 hours and 96 hours after editing by HSPCs treated with DMSO; 4 μM p38i; 4 μM ERKi or 2 μM JNKi prior to electroporation with HS RNP or HS / AAV6. [Figure 9] Figure 9. A) Schematic of the experimental design. B) Quantification of mitochondrial superoxide by MitoSOX in purified CD3+ T cells from two different donors, non-edited or edited, after treatment with 4 μM or 10 μM p38i 24 hours after editing. [Figure 10-1]Figure 10. A) Schematic of the experimental design. B) Percentage of p16+ senescent cells at 24 and 96 hours GE. (UT EL = cells that received electroporation only; HS / AAV6 = gene-edited cells, (n=1)). C) Percentage of SA-β-Gal+ senescent cells at 24 and 96 hours GE. Cells were transduced with increasing MOIs of AAV6 vector (2,000, 10,000, 20,000) and analyzed for both GFP- and GFP+ subfractions where indicated (n=1). D) Number of colonies formed by HSPCs treated with the indicated conditions at 24 and 96 hours after gene editing (n=3). E) Percentage of SA-β-Gal+ senescent cells in edited cell-derived colonies at 24 and 96 hours GE in GFP- and GFP+ subfractions (n=8). F,G,H) Percentage of human CD45+ cells in peripheral blood (PB) of NSG mice transplanted with edited HSPCs treated as indicated. I,J) Percentage of SA-β-Gal+ senescent cells among all human engrafted HSPCs (CD45+ cells, I) or BM-derived CD34+ cells (J) at endpoint (15 weeks). Cells were transduced with increasing MOIs of AAV6 vectors (2,000, 10,000, 20,000) and, where indicated, analysis was performed on both GFP- and GFP+ subfractions (I: n=4, 5, 5, 5, 3, 4, 4, 4, 5, 5; J: I: n=4, 5, 5, 5, 3, 15, 15, 4, 5, 5). Mann-Whitney test. *p<0.05, **p<0.01. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 11-1]Figure 11. A) Schematic of experimental design. B) Percentage of HDR-edited alleles by digital droplet PCR (ddPCR) (HS / AAV6 DMSO: n=1; HS / AAV6+ATMi: n=2). C) Percentage of p16+ senescent cells after 24 and 96 hours of GE (UT EL = cells only electroporated; HS / AAV6 = gene-edited cells, (n=1)). C) Percentage of GFP+ cells within HSPC subpopulations, ranging from more undifferentiated (CD90+) to more differentiated (CD133-) (n=11, 4). D) Relative expression of IL1A and IL6 in HS / AAV6-edited HSPCs 96 hours after electroporation in the presence or absence of ATMi (n=2). E) Number of colonies formed by HSPCs treated as indicated after 24 and 96 hours of GE. F) Percentage of human CD45+ cells in peripheral blood (PB) of NSG mice transplanted with edited HSPCs treated as indicated (n=25, 8). G) Percentage of human CD45+ cells in total BM from NSG mice transplanted with edited HSPCs 18 weeks after transplantation (n=7, 8). H) Percentage of SA-β-Gal+ senescent cells among BM-derived CD34+ cells at endpoint (18 weeks) (n=26, 20, 17, 17). Cells were edited ex-vivo in the presence or absence of p53 inhibition with GSE56. I, J, K) Relative expression of p21, p16, and IL8 in BM-derived HSPCs 18 weeks after transplantation in the indicated conditions (n=5). L) Number of colonies formed by BM-derived HSPCs (n=13, 14). Mann-Whitney test. *p<0.05, **p<0.01, ***p<0.001. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 12-1]Figure 12. A) Schematic of experimental design. B) Percentage of HDR-edited alleles by digital droplet PCR (ddPCR) (HS / AAV6: n = 20; HS / AAV6+ANAK: n = 15). C) Percentage of GFP+ cells within HSPC subpopulations, from more undifferentiated (CD90+) to more differentiated (CD133-) (n = 8, 9). D) Relative expression of CDKN1A (p21) in HS / AAV6-edited HSPCs 24 and 96 hours after electroporation (24 hours: n = 3, 2, 4, 5; 96 hours: n = 3, 2, 4, 3). E) Number of colonies formed by HSPCs treated with -DSB and HS / AAV6 in the presence or absence of anakinra, 24 and 96 hours after electroporation (24 hours: n = 7, 2, 5, 5; 96 hours: 3, 2, 5, 4). F) Culture composition of different subpopulations of edited HSPCs 24 and 96 hours after gene editing (24 hours: n=4, 2, 5, 3; 96 hours: n=4, 2, 6, 5). Mann-Whitney test. ns, p>0.05, *p<0.05. [Figure 12-2] This is a continuation of Figure 12-1. [Figure 13-1] Figure 13. A) GSEA for whole transcriptome analysis 24 and 96 hours after gene editing in the presence or absence of anakinra. B) Representative GSEA plots of two of the most upregulated inflammatory pathways from the Hallmark category. C) Relative expression of IL1A, IL8, and IL6 in HS / AAV6-edited HSPCs in the presence or absence of anakinra 96 ​​hours after electroporation (IL1A: n=9, 2, 9, 3; IL8: n=42, 2, 10, 4; IL6: n=27, 2, 6, 3;). Mann-Whitney test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 13-2] This is a continuation of Figure 13-1. [Figure 14-1]Figure 14. A) Quantification of immunofluorescence staining of nuclear NF-κB (>100 nuclei analyzed per sample) 24 and 96 hours after gene editing in the indicated conditions. B) Representative images of quantification in A (24 h: n=6, 6, 3, 3, 5, 6; 24 h: n=6, 6, 3, 3, 6, 4). Mann-Whitney test. ns, p>0.05, *p<0.05, **p<0.01. [Figure 14-2] This is a continuation of Figure 14-1. [Figure 15-1] Figure 15. A) Percentage of human CD45+ cells in peripheral blood (PB) of NSG mice transplanted with 3x105 HSPCs electroporated with HS RNP+AAV6 in the presence or absence of anakinra (ANAK). Mean + SEM (n=17, 28). B) Percentage of human edited cells in peripheral blood (PB) of NSG mice transplanted as shown in A (n=17, 28). C) Percentage of human CD45+ cells in bone marrow (BM) of NSG mice transplanted with 3x105 HSPCs electroporated with HS RNP+AAV6 in the presence or absence of anakinra (ANAK). Mean + SEM (n=16, 19). D) Percentage of human edited cells in bone marrow (BM) of NSG mice transplanted as shown in C (n=16, 19). E) Percentage of edited subpopulations (HSPCs, myeloid cells and B cells) in bone marrow (BM) of NSG mice transplanted as indicated in C (n=16, 19, 16, 19, 16, 19). F) Number of colonies formed by BM-derived CD34+ cells treated as in C (n=13, 20). G,H) Relative expression of IL8 (G, n=6) and CXCL10 (H, n=6) in HS / AAV6 BM-derived edited CD34+ cells with the indicated treatments. I) Percentage of p16+ senescent cells in BM-derived HSPCs (n=26, 20, 19, 17). J) Number of dominant unique BARs in human BM cells 5 weeks after transplantation into NSG mice (n=19, 15). K) Number of dominant unique BARs in human PB cells 8-9, 12 and 15 weeks after transplantation into NSG mice (n=6, 9, 6, 10, 6, 9). A, B: Linear mixed model (LME) at week 18. C–I: Mann-Whitney test; ns, p>0.05, *p<0.05, **p<0.01. [Figure 15-2]This is a continuation of Figure 15-1. [Figure 16-1] Figure 16. A) Schematic of the experimental design. B) Percentage of GFP+ cells within HSPC subpopulations, from more undifferentiated (CD90+) to more differentiated (CD133-) (n=8). C) Culture composition of different subpopulations of edited HSPCs after 24 and 96 hours of gene editing (n=6). D) Number of colonies formed by HSPCs treated with electroporation only (UT ELECTRO) and HS / AAV6 treated with or without anakinra 24 and 96 hours after electroporation (24 hours: n=7, 2, 5, 5; 96 hours: 3, 2, 5, 4). E) Percentage of p16+ senescent cells within GFP- and GFP+ subfractions after GE with or without anakinra 24 and 96 hours (24 hours: n=3; 96 hours: n= 3, 4, 4, 4, 4). F) Percentage of human CD45+ cells in the bone marrow (BM) of 15 week old mice transplanted with edited HSPCs as indicated (n=6, 5, 4). G) Percentage of human edited cells in the bone marrow (BM) of 15 week old NSG mice transplanted as indicated (n=5, 4). H) Percentage of p16+ senescent cells in BM-derived HSPCs treated as indicated (n=6, 5, 4). Mann-Whitney test; ns, p>0.05, *p<0.05. [Figure 16-2] This is a continuation of Figure 16-1. [Figure 17-1]Figure 17. A) Schematic of experimental design for one-hit IDLV gene editing. B) Schematic of experimental design for two-hit IDLV gene editing. C) Schematic of experimental design for AAV6 gene editing. D) Quantification of NBS1 positive cells from HSPCs edited with the indicated treatments and distribution of percentage of cells with bright points over time (n=5, 5, 2, 5). E) Quantification of immunofluorescence staining for nuclear NF-κB (>100 nuclei analyzed per sample) 24, 96 and 168 hours after gene editing in the indicated conditions (24 hours: n=2, 2, 2, 3, 3; 96 hours: n=3; 168 hours: n=3). F) Quantification of immunofluorescence staining for nuclear NF-κB (>100 nuclei analyzed per sample) 24 and 96 hours after gene editing in the indicated conditions (24 hours: n=2; 96 hours: n=1). G) Number of colonies formed by treated HSPCs 24 and 96 hours after gene editing in the indicated conditions (n=2). H) Percentage of GFP+ cells within HSPC subpopulations (n=5), ranging from more undifferentiated (CD90+) to more differentiated (CD133-). I) Apoptosis analysis performed on HSPC subfractions from H at GE24 hours. Early apoptosis: Annexin V+, 7AAD-; late apoptosis: Annexin V+, 7AAD+; necrosis: Annexin V-; 7AAD+ (n=5). J) Culture composition of different subpopulations of edited HSPCs 24 and 96 hours after gene editing (n=5). Mann-Whitney test. ns, p>0.05, *p<0.05, **p<0.01. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18-1]Figure 18. A) Schematic of experimental design. B) Percentage of CB-derived edited alleles by HDR by digital droplet PCR (ddPCR) (n=20, 11, 3,5). C) Relative expression of IL8, IL6 and CCL2 in HSPCs edited in the presence or absence of SC-514 or a combination of GSE56 and SC-514 (n=2). D) Number of colonies formed by HSPCs treated after 96 hours of GE (n=16, 3, 3, 3). E) Percentage of GFP+ cells within HSPC subpopulations, from more undifferentiated (CD90+) to more differentiated (CD133-) (n=2, 1). F) Number of colonies formed by HSPCs treated after 24 and 96 hours of GE (n=3). G) Percentage of p16+ senescent cells within GFP- and GFP+ subfractions at 24 and 96 h of GE in the presence or absence of SC514 (24 h: n=3; 96 h: n=3, 4, 4, 3, 3). Kruskal-Wallis test. ns, p>0.05, *p<0.05. [Figure 18-2] This is a continuation of Figure 18-1. [Figure 19-1]Figure 19. A) Percentage of human CD45+ cells in peripheral blood (PB) of NSG mice transplanted with 1.5x105 HSPCs electroporated with HS RNP+AAV6 in the presence of GSE56, SC-514 or GSE56+SC-514 (n=5). B) Percentage of human CD45+ cells 15 weeks after transplantation in bone marrow (BM) of NSG mice transplanted with HSPCs treated as indicated (n=8, 12, 9, 11). C) Number of colonies formed by 15 weeks after transplantation BM-derived HSPCs treated as indicated (n=6). D) Number of dominant unique BARs in human BM-derived cells at the end of the experiment (15 weeks) (n=19, 12, 16, 11). E) Number of unique indels obtained by on-target AAVS1 sequencing in human bone marrow cells of mice treated as indicated (n=5, 12, 6, 11). F, G) p16 intracellular levels (F, n=26, 20, 12, 12, 15, 14, 13, 13) and percentage of SA-β-Gal senescent cells (G, n=22, 22, 11, 11, 5, 5, 5, 5) in BM-derived CD34+ cells at endpoint (week 15). A: Linear mixed model (LME) at week 15. B-E: Kruskal-Wallis test. F-G: Mann-Whitney test; ns, p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 19-2] This is a continuation of Figure 19-1. [Figure 20-1]FIG. 20. A) Schematic of the experimental design: HSPCs were treated with DMSO or 4 μM p38i on days 1 and 2 after thawing and analyzed on days 1, 3, and 7. B) Quantification of Phospho-p38 in HSPCs at the indicated time points (n=5). C) Quantification of single- and double-stranded DNA breaks by alkaline comet assay on days 3 and 7 after thawing in DMSO- or p38i-treated cells (n=2, more than 100 cells analyzed for each condition). D) Representative confocal images of γH2AX (green) and pRPA (red) puncta and magnifications within the photographs. Nuclei were stained with DAPI. E) Quantification of γH2AX puncta on days 3 and 7 (n=8, 3, 7, 4). F) Quantification of pRPA puncta on days 3 and 7 (n=7, 3, 7, 3). G,H) Quantification of cytosolic ROS (G) detected by CM-H2DCFDA and mitochondrial superoxide (H) by MitoSOX in thawed day 3 and day 7 HSPCs (G: n=3, 3, 5, 5, 5, 4; H: n=3, 3, 6, 6, 5, 4). Mann-Whitney test. *p<0.05; **p<0.01. [Figure 20-2] This is a continuation of Figure 20-1. [Figure 20-3] Continuation of Figure 20-2. [Figure 21-1]FIG. 21. A) Schematic of experimental design: HSPCs were treated with DMSO, or 4 μM or 8 μM p38i on days 1 and 2 of thawing and electroporated with HS RNP or HS RNP+AAV6 on day 3. Subsequent analysis was performed 24 or 96 hours after electroporation. B) Percentage of edited allele in CD34+ cells upon DMSO or p38i treatment measured by ddPCR (n=10, 10, 3). C) Number of colonies formed by HSPCs treated with DMSO, 4 μM or 8 μM p38i 24 hours after electroporation (+DSB(HS): n=7, 7, 3; HS / AAV6: n=10, 10, 5). D, E) Quantification of cytosolic ROS (D) detected by CM-H2DCFDA and mitochondrial superoxide (E) by MitoSOX in HS / AAV6-edited HSPCs 24 and 96 hours after electroporation (D: n=3, 4, 3, 3; E: n=2). F) Expression of phosphorylated p38-MAPK in different subpopulations expressed as median fluorescence intensity (MFI) at the indicated time points after gene editing. G) Percentage of edited allele in CD34+CD133+CD45RA-CD90+ cells upon DMSO or p38i treatment measured by ddPC (n=3). C: Kruskal-Wallis test. A-G: Mann-Whitney test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Figure 21-2] This is a continuation of Figure 21-1. [Figure 22-1]Figure 22. A) Schematic of the experimental design: HSPCs were treated with DMSO or 4 μM p38 on days 1 and 2 after thawing, then electroporated with HS RNP or HS RNP+AAV6 on day 3. Transplantation into NSG mice was performed 24 h after electroporation. B, C) Percentage of human CD45+ cells in peripheral blood (PB) (B) and bone marrow (BM) (C) of NSG mice transplanted with 1–1.5 × 105 HSPCs pretreated with DMSO or 4 μM p38i and then electroporated with HS RNP or HS RNP+AAV6. B: Mean ± SEM; C: Median ± SEM; (n=7, 10, 9, 8). D) Number of dominant unique BARs in human BM-derived cells at the end of the experiment (15 weeks). Median; (n=9, 8). E) Peripheral blood composition: percentage of CD19+, CD13+ or CD3+ cells within the human CD45+ population (n=7, 10, 9, 8). F) Schematic of experimental design: BM-derived CD34+ cells were stimulated with CFU-C in an in vitro assay at endpoint. G) Number of colonies formed by CD34+ purified from BM at week 15 (n=7, 10, 9, 8 from two independent experiments). H) Percentage of fluorescent SA-β-galactosidase positive cells measured in BM-derived CD34+ at week 15 (n=5, 3, 5, 2). Mann-Whitney test, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. [Figure 22-2] This is a continuation of Figure 22-1. [Figure 23-1]Figure 23. A) Schematic of experimental design: HSPCs were treated with DMSO or 4 μM p38i on days 1 and 2 after thawing, then electroporated with HS RNP+AAV6 on day 3 (where indicated). 24 hours after electroporation, HSC / MPP pools (CD34+CD133+CD45RA-CD90+) were sorted and seeded as single cells in differentiation medium. B) Percentage of mono-, bi- and multi-lineage colonies were calculated based on marker expression and representation within single colonies. C) Percentage of colonies with indicated lineage composition. (B, C: n=4; >500 colonies were analyzed for each donor and each condition). D) Schematic of experimental design: HSPCs were treated with DMSO or 4 μM p38i on days 1 and 2 after thawing, then electroporated with HS RNP+AAV6 on day 3. 24 hours after electroporation, HSC / MPP pools (CD34+CD133+CD45RA-CD90+GFP+ or GFP-) were sorted and processed for single-cell RNA-sequencing analysis. E) Uniform Manifold Approximation and Projection (Umap) plot containing scRNA-seq data. Clusters and associated cell types are labeled and colored as indicated. F) Stacked bar plot showing the distribution of identified clusters among samples. G) Heatmap showing LogFC values ​​of differentially expressed genes (DEGs) among the indicated samples. [Figure 23-2] This is a continuation of Figure 23-1. [Figure 24]FIG. 24. A) Schematic of experimental design: HSPCs were treated with DMSO, 2 μM JNKi, or 4 μM ERKi on days 1 and 2 after thawing and electroporated with HS RNP or HS RNP+AAV6 on day 3. B) GFP expression (n=2, 2, 1) in different edited HSPC subpopulations (CD34+CD133-; CD34+CD133+ and CD34+CD133+CD90+ cells) with the indicated treatments (DMSO; 2 μM JNKi; 4 μM ERKi) 96 hours after editing. C) Number of colonies formed 24 and 96 hours after editing by HSPCs treated with DMSO, 2 μM JNKi, or 4 μM ERKi prior to electroporation with HS RNP or HS / AAV6. [Figure 25-1]Figure 25. A) Schematic of experimental design: PBMC-derived CD4+ T cells were treated with DMSO or 10 μM p38i on days 1 and 2 after purification and, where indicated, electroporated with HS RNP+AAV6 on day 3. B) Percentage of edited cells measured by flow cytometry as dNGFR+ cells (n=3). C) Relative quantification of percentages of T cell subpopulation composition within CD4+ T cells (TEMRA: CD62L-CD45RA+; CM: CD62L+CD45RA-; EM: CD62L-CD45RA-; TSCM: CD62L+CD45RA+) 96 hours after electroporation. D) Apoptosis analysis performed 96 hours after electroporation within total CD4+ T cells. Early apoptosis: Annexin V+, 7AAD-; Late apoptosis: Annexin V+, 7AAD+; Necrosis: Annexin V-; 7AAD+. E) CD4+ T cell growth curves. Cells from the reported conditions were counted at the indicated time points and the total number of cells and the fold increase between untreated (-) and p38 inhibitor cells (p38i) are reported graphically. F) Quantification of mitochondrial superoxide by MitoSOX in purified CD4+ T cells from two different donors that were unedited or edited after treatment with 10 μM p38i 24 h after editing (n=2). G,H) Percentage of senescent cells assessed by SPiDER SA-β-galactosidase (G) or p16 intracellular staining (H) at days 1, 13 and 20 after gene editing (n=3). Mann-Whitney test. *p<0.05. [Figure 25-2] This is a continuation of Figure 25-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0166] Detailed Description of the Invention Cell survival and engraftment In one aspect, the invention provides the use of one or more senescence inhibitors to increase survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0167] In a further aspect, the present invention provides one or more senescence inhibitors for use in hematopoietic cell gene therapy, hematopoietic stem cell gene therapy, hematopoietic progenitor cell gene therapy and / or T cell gene therapy.

[0168] In a further aspect, the present invention provides one or more senescence inhibitors for use in increasing survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0169] In some embodiments, the use is an in vitro or ex vivo use.

[0170] In some embodiments, the gene therapy is hematopoietic cell gene therapy, hematopoietic stem cell gene therapy and / or hematopoietic progenitor cell gene therapy.

[0171] In some embodiments, the cells are hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells.

[0172] Current protocols for gene transfer and editing require long-term ex-vivo culture, high viral vector doses, and nuclease-induced DNA DSBs that activate DNA damage response (DDR) pathways leading to cell cycle arrest. Emerging data show that cellular detection of viral vectors, as employed in classical gene therapy settings, unexpectedly also induces DDR, instead of triggering innate immune-mediated recognition of viral nucleic acids or proteins.

[0173] The DDR pathway is an evolutionarily conserved set of actions that converge on key decision-makers, such as the tumor suppressor p53, to enforce cell cycle arrest (Piras, F. et al., 2017, EMBO Mol Med 9: 1198-1211). Activation of the DDR pathway leads to DDR-dependent inflammation (Di Micco, R., 2017, Trends Mol. Med. 23: 1067-1070). We have previously demonstrated that activation of the DDR pathway impairs hematopoietic reconstitution of genetically modified cells upon transplantation (Schiroli, G. et al., 2019, Cell Stem Cell 24: 551-565; and Conti, A. & Di Micco, R., 2018, Genome Med 10: 66).

[0174] In some embodiments, the SASP inhibitor (e.g., an IL-1 inhibitor and / or an NF-κB inhibitor) inhibits DDR-dependent inflammation. Advantageously, inhibition of DDR-dependent inflammation increases survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0175] Prolonged DDR signaling is causally linked to the establishment of cellular senescence, a state in which cells are still alive but cannot proliferate any further. Accumulation of the cell cycle inhibitors p21 and p16 is associated with senescence. Senescent cells are also characterized, for example, by a senescence-associated secretory phenotype (SASP). Through the SASP, which is mainly characterized by inflammatory cytokines, senescent cells may exert deleterious paracrine functions on bystander cells.

[0176] In some embodiments, the one or more senescence inhibitors inhibit a cellular senescence program. Suitably, inhibition of the cellular senescence program increases survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0177] As used herein, the term "survival" refers to the ability of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells to remain viable (e.g., not die or undergo apoptosis) during in vitro or ex vivo culture. Hematopoietic stem and / or progenitor cells and / or T cells may undergo high apoptosis after, for example, being transduced with a viral vector during cell culture, and thus, surviving cells may have avoided apoptosis and / or cell death.

[0178] Cell survival can be easily analyzed by those skilled in the art. For example, the number of live, dead and / or apoptotic cells in a cell culture can be quantified at the beginning of the culture and / or after a certain period of culture (e.g., about 6 or 12 hours, or 1, 2, 3, 4, 5, 6, 7 or more days; preferably, the period begins with the transduction of the cells with the viral vector). The effect of the inhibitor according to the invention on cell survival can be evaluated by comparing the number and / or percentage of live, dead and / or apoptotic cells in the presence and absence of the inhibitor at the beginning and / or end of the culture period, under otherwise substantially identical conditions.

[0179] The number and / or percentage of cells (e.g., live, dead and / or apoptotic cells) in a particular case can be quantified using any of several methods known in the art, including the use of a hemocytometer, an automated cell counter, a flow cytometer and a fluorescence activated cell sorter. These techniques are capable of distinguishing between live, dead and / or apoptotic cells. Additionally or alternatively, apoptotic cells can be quantified using readily available apoptosis assays (e.g., assays based on the detection of phosphatidylserine (PS) on the cell membrane surface, such as by the use of Annexin V, which binds to exposed PS; apoptotic cells can be quantified by the use of fluorescently labeled Annexin V, which can also be used to complement other techniques.

[0180] As used herein, the term "engraftment" refers to the ability of hematopoietic stem and / or progenitor cells and / or T cells to establish and survive within a subject following their transplantation, i.e., short-term and / or long-term following transplantation. For example, engraftment can refer to the number and / or percentage of hematopoietic cells and / or T cells (e.g., graft-derived cells) lineage-derived from transplanted hematopoietic stem and / or T cells detected about 1 day to 24 weeks, 1 day to 10 weeks, or 1 to 30 days, or 10 to 30 days following transplantation. In a xenotransplantation model of human hematopoietic stem and / or progenitor cell engraftment and repopulation, engraftment can be assessed in peripheral blood, for example, as the percentage of cells derived from the human xenograft (e.g., CD45 surface marker positive). In one embodiment, engraftment is assessed about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days following transplantation. In another embodiment, engraftment is assessed at about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks post-transplant. In another embodiment, engraftment is assessed at about 16-24 weeks, preferably 20 weeks, post-transplant.

[0181] Engraftment can be easily analyzed by one of skill in the art. For example, transplanted hematopoietic stem and / or progenitor cells and / or T cells can be engineered to contain markers (e.g., reporter proteins such as fluorescent proteins) that can be used to quantitate graft-derived cells. Samples for analysis can be extracted from the relevant tissue and analyzed ex vivo (e.g., using flow cytometry).

[0182] Suitably, the inhibitor for use according to the invention may improve engraftment of gene-edited hematopoietic stem and / or progenitor cells and / or T cells compared to gene editing without the use of an inhibitor. Suitably, engraftment at a given time point may be increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to engraftment of untreated gene-edited hematopoietic stem and / or progenitor cells and / or untreated gene-edited T cells.

[0183] Suitably, the inhibitor for use according to the invention may improve the engraftment of transduced hematopoietic stem and / or progenitor cells and / or T cells compared to transduction without the use of the inhibitor. Suitably, engraftment at a given time point may be increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to the engraftment of untreated transduced hematopoietic stem and / or progenitor cells and / or untreated transduced T cells.

[0184] In a preferred embodiment, the inhibitor (or inhibitors) for use according to the invention do not adversely affect proliferation of gene edited and / or transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells compared to untreated gene edited hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells.

[0185] Gene editing efficiency In a further aspect, the present invention provides the use of one or more senescence inhibitors to increase gene editing efficiency of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0186] In a further aspect, the present invention provides one or more senescence inhibitors for use in hematopoietic cell gene therapy, hematopoietic stem cell gene therapy, hematopoietic progenitor cell gene therapy and / or T cell gene therapy. Suitably, the one or more senescence inhibitors increase the efficiency of gene editing of said cells.

[0187] In some embodiments, the IL-1 inhibitor and / or the NF-κB inhibitor inhibits DDR-dependent inflammation. Suitably, inhibition of DDR-dependent inflammation increases gene editing efficiency of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0188] In some embodiments, the senescence inhibitor (e.g., a MAPK inhibitor) inhibits a cellular senescence program. Suitably, inhibition of the cellular senescence program increases gene editing efficiency of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0189] In some embodiments, the use is an in vitro or ex vivo use.

[0190] In some embodiments, the gene therapy is hematopoietic cell gene therapy, hematopoietic stem cell gene therapy and / or hematopoietic progenitor cell gene therapy.

[0191] In some embodiments, the cells are hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells.

[0192] Increasing gene editing efficiency may refer to increasing gene editing of cells (e.g., hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells) using an inhibitor (or combination of inhibitors) according to the invention compared to gene editing achieved under otherwise substantially identical conditions in the absence of the inhibitor. Thus, when using a viral vector to introduce a gene editing mechanism, increasing efficiency may allow for a reduction in the multiplicity of infection (MOI) and / or time required to achieve effective transduction.

[0193] In one embodiment, the percentage of edited cells is increased. Methods for determining the percentage of edited cells are known in the art. Suitable methods include flow cytometry, fluorescence activated cell sorting (FACS) and fluorescence microscopy. The techniques employed are preferably amenable to automation and / or high throughput screening.

[0194] For example, a cell population can be edited using a vector carrying a reporter gene. Preferably, the reporter gene can be expressed when the cells are edited. Suitable reporter genes include genes encoding fluorescent proteins, such as green, yellow, cherry, cyan or orange fluorescent proteins. Once the cell population has been edited, both cells that express and those that do not express the reporter gene can be quantified using a suitable technique, such as FACS. The percentage of edited cells can then be calculated.

[0195] Alternatively, quantitative PCR (qPCR) can be used to determine the percentage of gene-edited cells without the use of a reporter gene. For example, cells (e.g., CD34 + Single colonies of 1000 ng / ml (cells) can be picked from the semi-solid culture and each colony can be subjected to individual qPCR to determine the percentage of positive gene-edited colonies among those analyzed.

[0196] Methods for determining vector copy number are also known in the art. The techniques employed are preferably amenable to automation and / or high-throughput screening. Suitable techniques include quantitative PCR (qPCR) and Southern blot-based approaches.

[0197] Increased gene editing efficiency may refer to increasing the number of cells (e.g., hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells) in which a target gene or site has been edited (e.g., disrupted, replaced, deleted, or has a nucleic acid sequence inserted therein or therein) in an intended manner after transduction of a cell population with a viral vector using an inhibitor (or combination of inhibitors) according to the present invention, compared to that achieved under otherwise substantially identical conditions in the absence of the inhibitor. Thus, increased gene editing efficiency may allow for a reduction in the multiplicity of infection (MOI) and / or transduction time required to achieve effective gene editing. Methods for determining whether a target gene or site has been edited are known in the art.

[0198] Increased gene editing efficiency can refer to an increase in fitness of a gene-edited cell (e.g., a hematopoietic cell, a hematopoietic stem cell, a hematopoietic progenitor cell and / or a T cell) edited with an inhibitor (or a combination of inhibitors) according to the invention compared to that achieved under otherwise substantially identical conditions in the absence of the inhibitor.

[0199] An increase in gene editing efficiency can refer to an increase in viability of a gene-edited cell (e.g., a hematopoietic cell, a hematopoietic stem cell, a hematopoietic progenitor cell and / or a T cell) edited with an inhibitor (or a combination of inhibitors) according to the invention compared to that achieved under otherwise substantially identical conditions in the absence of the inhibitor.

[0200] For gene editing, e.g., using the CRISPR / Cas system, preferably the vector used to transduce the cell population is a non-integrating vector (e.g., an integration-defective lentiviral vector, IDLV).

[0201] In one embodiment, the inhibitor (or combination of inhibitors) for use according to the present invention improves gene editing efficiency compared to gene editing without a drug (i.e. standard gene editing). Suitably, gene editing efficiency may be improved by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 3-fold or more.

[0202] In one embodiment, the inhibitor (or combination of inhibitors) for use according to the present invention increases gene editing efficiency compared to gene editing without drugs (i.e. standard gene editing). Preferably, the percentage of edited cells is increased. Preferably, the percentage of edited cells can be increased by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300% or more. Preferably, the percentage of edited cells can be increased by 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0203] Advantageously, gene editing efficiency may be improved in specific cell compartments. Advantageously, gene editing is improved in the undifferentiated HSPC cell compartment. Advantageously, gene editing is improved in the CD34 + CD133 -Preferably, gene editing is performed to improve the expression of CD34 + CD133 + Preferably, gene editing is performed to improve the expression of CD34 + CD133 + CD90 + It can be improved in cells.

[0204] Preferably, CD34 + CD133 + CD90 + The gene editing efficiency of a cell may be improved by at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 1.8 fold, 1.9 fold, 2.0 fold, 2.1 fold, 2.2 fold, 2.3 fold, 2.4 fold, 2.5 fold, 3 fold or more.

[0205] Methods for gene editing of cell populations In one aspect, the invention provides a method for gene editing of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells, comprising: (a) introducing a gene editing mechanism into a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells using one or more vectors; and (b) editing the genome of the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells. contacting the cell population with one or more senescence inhibitors prior to, simultaneously with, or after introducing the gene editing mechanism into the cell population.

[0206] In a further aspect, the invention provides a method for gene editing of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells, comprising: (a) contacting a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells with one or more senescence inhibitors; (b) introducing gene editing machinery into a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells using one or more vectors; and (c) editing the genome of said hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells. The present invention provides a method comprising:

[0207] In a further aspect, the invention provides a method for gene editing of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells, comprising: (a) introducing a gene editing mechanism into a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells using one or more vectors; (b) contacting the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more senescence inhibitors; and (c) editing the genome of said hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells. The present invention provides a method comprising:

[0208] In a further aspect, the invention provides a method for gene editing of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells, comprising: (a) introducing a gene editing mechanism into a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells using one or more vectors and simultaneously contacting the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells with one or more senescence inhibitors; and (b) editing the genome of said hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells. The present invention provides a method comprising:

[0209] In some embodiments, the method is a method of gene editing a population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells.

[0210] In some embodiments, the steps of introducing a gene editing mechanism into the cell population and contacting the cell population with one or more senescence inhibitors are performed ex vivo or in vitro.

[0211] In some embodiments, the population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells is contacted with a MAPK / ERK signaling inhibitor (e.g., a MAPK inhibitor) prior to or simultaneously with the step of introducing a gene editing mechanism into the cells, preferably prior to the step of introducing a gene editing mechanism into the cells.

[0212] In some embodiments, a population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells is contacted with an IL-1 inhibitor and / or an NF-κB inhibitor prior to, simultaneously with or after introducing a gene editing mechanism into the cells.

[0213] Thus, in one embodiment of the method for gene editing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells, steps (a) and (b) can be performed simultaneously, while in another embodiment, steps (a) and (b) can be performed sequentially, either with step (a) being performed before step (b), or with step (b) being performed before step (a).

[0214] In one embodiment, the gene editing machinery may comprise a nuclease, such as a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, or a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system.

[0215] The gene editing mechanism (e.g., a CRISPR / Cas system) may include one or more guide RNAs complementary to at least one target gene in a cell, an RNA-dependent DNA endonuclease enzyme, or a nucleotide sequence encoding the endonuclease (e.g., a nucleotide sequence encoding a Cas9 protein or enCas9). In one embodiment, the gene editing mechanism may be a CRISPR / Cas system.

[0216] In one embodiment, the gene editing mechanism may be provided by one or more nucleotide sequences. Preferably, the nucleotide sequences encoding the gene editing mechanism may be introduced into the cell sequentially or simultaneously. In one embodiment, the inhibitor may be contacted with the cell at the same time as the gene editing mechanism is introduced into the cell. In one embodiment, the one or more nucleotide sequences encoding the gene editing mechanism are introduced into the cell by electroporation. In one embodiment, the one or more nucleotide sequences are introduced into the cell by transduction. Preferably, the nucleotide sequences may be introduced by transduction of a viral vector. For example, the Cas9 ribonucleoprotein may be introduced into the cell by electroporation prior to AAV6 transduction for delivery of the donor DNA template.

[0217] As used herein, the term "introducing" refers to a method for inserting foreign DNA or RNA into a cell. As used herein, the term "introducing" includes both transduction and transfection methods. Transfection is a method of introducing nucleic acid into a cell by a non-viral method. Transduction is a method of introducing foreign DNA or RNA into a cell via a viral vector.

[0218] In one embodiment, AAV transduction is used to deliver the donor DNA template.

[0219] In one embodiment, AAV6 transduction is used to deliver the donor DNA template.

[0220] Methods for transducing cell populations In a further aspect, the invention provides a method of transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with a viral vector, comprising transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more viral vectors, contacting the cell population with one or more senescence inhibitors before, simultaneously with or after transducing the cell population.

[0221] In a further aspect, the invention provides a method for transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with a viral vector, comprising: (a) contacting a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more senescence inhibitors; and (b) transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more viral vectors. The present invention provides a method comprising:

[0222] In a further aspect, the invention provides a method for transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with a viral vector, comprising: (a) transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more viral vectors; and (b) contacting the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more senescence inhibitors. The present invention provides a method comprising:

[0223] In a further aspect, the invention provides a method of transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with a viral vector, comprising contacting the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more senescence inhibitors while simultaneously transducing the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with the one or more viral vectors.

[0224] In some embodiments, the method is a method of gene editing a population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells.

[0225] In some embodiments, steps (a) and (b) are performed ex vivo or in vitro.

[0226] In some embodiments, the population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells is contacted with a MAPK / ERK signaling inhibitor (e.g., a MAPK inhibitor) prior to or simultaneously with transducing the cells, preferably prior to transducing the cells.

[0227] In some embodiments, a population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells is contacted with an IL-1 inhibitor and / or an NF-κB inhibitor prior to, simultaneously with or after transducing the cells.

[0228] Hematopoietic Stem and Progenitor Cells Stem cells can differentiate into many cell types. Cells capable of differentiating into all cell types are known as totipotent. In mammals, only zygotes and early embryonic cells are totipotent. Stem cells are found in most, if not all, multicellular organisms. Stem cells are characterized by their ability to renew themselves by cell division and to differentiate into a diverse range of specialized cell types. The two broad types of mammalian stem cells are embryonic stem cells, which are isolated from the inner cell mass of blastocysts, and adult stem cells, which are found in adult tissues. In the developing embryo, stem cells can differentiate into all of the specialized embryonic tissues. In the adult organism, stem and progenitor cells act as the body's repair system, not only replenishing specialized cells but also maintaining the normal turnover of regenerative organs such as blood, skin or intestinal tissues.

[0229] Hematopoietic stem cells (HSCs) are multipotent stem cells that can be found, for example, in peripheral blood, bone marrow, and umbilical cord blood. HSCs have the capacity for self-renewal and differentiation into any blood cell lineage. HSCs can repopulate the entire immune system, as well as the erythroid and myeloid lineages of all hematopoietic tissues (e.g., bone marrow, spleen, and thymus). HSCs provide lifelong production of all hematopoietic cell lineages.

[0230] Hematopoietic progenitor cells have the capacity to differentiate into specific types of cells. However, in contrast to stem cells, hematopoietic progenitor cells are already quite specific and tasked with differentiating into their "target" cells. The difference between stem cells and progenitor cells is that stem cells can proliferate indefinitely, whereas progenitor cells can only divide a limited number of times. Hematopoietic progenitor cells can only be strictly distinguished from HSCs by functional in vivo assays (i.e., transplantation and demonstration of their ability to give rise to all blood lineages over the long term).

[0231] The hematopoietic stem and progenitor cells of the present invention contain the CD34 cell surface marker (CD34 + (It is written as ".")

[0232] In one embodiment, the cells used in the present invention are hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells transduced with one or more viral vectors.

[0233] In one embodiment, the cells for use in the present invention are HSPCs.

[0234] In one embodiment, the cells for use in the present invention are primitive HSPCs. In one embodiment, the primitive subset of HSPCs expresses CD90 + In one embodiment, the primitive subset of HSPCs refers to a population of HSCs that are CD34 + CD133 + and CD90 + It refers to a population of cells that are

[0235] In one embodiment, the cells for use in the present invention are HSCs.

[0236] Hematopoietic Stem and / or Progenitor Cell (HSPC) Source The population of hematopoietic stem and / or progenitor cells may be obtained from a tissue sample.

[0237] For example, populations of hematopoietic stem and / or progenitor cells may be obtained from peripheral blood (e.g., adult and fetal peripheral blood), umbilical cord blood, bone marrow, liver or spleen. Preferably, the cells are obtained from peripheral blood or bone marrow. The cells may be obtained after mobilization of the cells in vivo by means of growth factor treatment.

[0238] Mobilization may be achieved, for example, with G-CSF, plerixafor, or a combination thereof. Other drugs such as NSAIDs and dipeptidyl peptidase inhibitors may also be useful as mobilizing agents.

[0239] With the availability of the stem cell growth factors GM-CSF and G-CSF, most hematopoietic stem cell transplant procedures are now performed using stem cells harvested from peripheral blood rather than bone marrow. Harvesting peripheral blood stem cells provides a larger number of recipients, does not require the donor to undergo general anesthesia to harvest the recipients, and may result in shorter engraftment times and lower long-term relapse rates.

[0240] Bone marrow may be harvested by standard aspiration techniques (steady state or post-mobilization) or by next generation harvesting tools (eg, the Marrow Miner).

[0241] In addition, hematopoietic stem and progenitor cells may also be derived from induced pluripotent stem cells.

[0242] Characteristics of HSC HSCs generally have a low forward and side scatter profile by flow cytometry. Some are metabolically quiescent, as indicated by rhodamine labeling, which allows for the determination of mitochondrial activity. HSCs may contain specific cell surface markers, such as CD34, CD45, CD133, CD90, and CD49f. HSCs can also be defined as cells that lack expression of CD38 and CD45RA cell surface markers. However, the expression of some of these markers depends on the developmental stage and tissue-specific context of the HSC. Some HSCs, referred to as "side population cells," exclude Hoechst33342 dye when detected by flow cytometry. Thus, HSCs have classification properties that allow for their identification and isolation.

[0243] Negative markers CD38 is the most established and useful single negative marker for human HSCs.

[0244] Human HSCs can also be negative for lineage markers such as CD2, CD3, CD14, CD16, CD19, CD20, CD24, CD36, CD56, CD66b, CD271, and CD45RA, however, these markers may have to be used in combination for HSC enrichment.

[0245] "Negative markers" should be understood to refer to the lack of expression of these markers in human HSCs.

[0246] Positive markers CD34 and CD133 are the most useful positive markers for HSCs.

[0247] Some HSCs are also positive for lineage markers such as CD90, CD49f and CD93, however, these markers may have to be used in combination for HSC enrichment.

[0248] "Positive markers" should be understood to mean that human HSCs express these markers.

[0249] In one embodiment, the hematopoietic stem and progenitor cells are CD34 + CD38 - It is a cell.

[0250] differentiated cells Differentiated cells are more specialized cells compared to stem or progenitor cells. Differentiation occurs during the development of multicellular organisms as the organism changes from a single zygote to a complex system of tissues and cell types. Differentiation is also a common process in adults: adult stem cells divide to generate fully differentiated daughter cells during tissue repair and normal cell turnover. Differentiation dramatically changes cell size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are primarily due to highly regulated modifications in gene expression. That is, differentiated cells are cells that have specific structures and perform specific functions resulting from developmental processes, which involve the activation and inactivation of specific genes. Here, differentiated cells include differentiated cells of the hematopoietic lineage, such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells, T cells, B cells, and NK cells. For example, differentiated cells of the hematopoietic lineage can be distinguished from stem and progenitor cells by detection of cell surface molecules that are not expressed or are expressed poorly on undifferentiated cells. Examples of suitable human cell lineage markers include CD33, CD13, CD14, CD15 (myeloid), CD19, CD20, CD22, CD79a (B), CD36, CD71, CD235a (erythroid), CD2, CD3, CD4, CD8 (T), CD56 (NK).

[0251] In one embodiment, the hematopoietic cells referred to herein are T cells.

[0252] T cells T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of TCR on their cell surface. There are various types of T cells, as summarized below.

[0253] Cytolytic T cells (TC cells, or CTLs) destroy virus-infected and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of all nucleated cells. CD8 cells are activated by IL-10, adenosine, and other molecules secreted by regulatory T cells. + The cells are inactivated and rendered anergic, preventing autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0254] Regulatory T cells (Treg cells), previously known as suppressor T cells, are crucial for maintaining immune tolerance: their main role is to shut down T cell-mediated immunity at the end of an immune response and to suppress autoreactive T cells that escape the negative selection process in the thymus.

[0255] Natural Treg cells (CD4 + CD25 + FoxP3 + Treg cells (also known as Treg cells) arise in the thymus and express both developing T cells and myeloid lineages activated by TSLP (CD11c + ) and plasma cell lines (CD123 + ) have been implicated in interactions with dendritic cells. Natural Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can disrupt the development of regulatory T cells and lead to the fatal autoimmune disease IPEX.

[0256] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during the course of a normal immune response.

[0257] Helper T cells (TH cells) assist other white blood cells in immunological processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when peptide antigens are presented by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9, or THF, and secrete different cytokines to promote different types of immune responses.

[0258] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. Memory T cells rapidly expand to large numbers of effector T cells when re-exposed to their cognate antigen, thus providing the immune system with a "memory" of past infection. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells are classified as CD4 + or CD8 + Memory T cells generally express the cell surface protein CD45RO.

[0259] CD4 + Two major classes of Treg cells have been described: innate and adaptive Treg cells.

[0260] Natural killer T cells (NKT cells) are a subset of D1d-restricted T cells at the interface of the innate and adaptive immune systems. NKT cells recognize lipids and glycolipids presented by CD1d molecules, members of the CD1 family of antigen-presenting molecules, rather than peptide / MHC complexes. Natural NKT cells co-express the αβ TCR and various molecular markers commonly associated with NK cells, including NK1.1, CD16 and CD56 expression, and granzyme production. Thus, these cells possess characteristics of both conventional T cells and NK cells, with the exception of NK1.1. + Cells and NK1.1 -Both cells, as well as CD4 + , CD4 - , CD8 + and CD8 - Contains cells.

[0261] NKT cells can be subdivided into functional subsets that rapidly respond to a wide range of glycolipids and stress-related proteins using T cell- or natural killer (NK) cell-like effector mechanisms. NKT cells are also considered key players in tumor immune surveillance, as they primarily mediate effects on immune responses via the secretion of cytokines.

[0262] The cells according to the invention may be of any of the cell types mentioned above.

[0263] For example, T cells or NK cells can be activated and / or expanded by treatment with an anti-CD3 monoclonal antibody, for example, before being transduced and / or edited as described above.

[0264] Alternatively, the cells may be derived from ex vivo differentiation of induced or embryonic precursor cells into T cells. Alternatively, immortalized T cell lines that retain their lytic function may be used.

[0265] In one embodiment, cells for use in the present invention are T cells transduced with one or more viral vectors.

[0266] Aging inhibitors Senescence is a process in which cells permanently stop dividing but are still metabolically active and do not die. Thus, senescent cells can gradually accumulate in a cell population. Cellular senescence is caused by ageing and severe DNA damage. As mentioned above, prolonged DDR signaling is causally linked to the establishment of cellular senescence, which also results in the activation of the senescence-associated secretory phenotype (SASP).

[0267] Senescence inhibitors can be divided into two main categories: (i) senescence inhibitors (e.g., MAPK / ERK signaling pathway inhibitors, e.g., p38 inhibitors), which are a class of drugs that slow down the rate at which senescent cells form, and (ii) SASP inhibitors (e.g., IL-1 inhibitors and NF-κB inhibitors), which are inhibitors of senescence-associated inflammation. Thus, senescence inhibitors act before the formation of senescent cells, and SASP inhibitors act downstream (i.e., after senescent cells have formed) to inhibit the senescence-related phenotype (i.e., SASP). Thus, senescence inhibitors and SASP inhibitors are expected to act before, during, and after gene editing and / or transduction of cells. Furthermore, senescence inhibitors are desirably added before gene editing and / or transduction of cells, while SASP inhibitors are preferably added during and / or after gene editing and / or transduction of cells.

[0268] In some embodiments, the inhibitor of senescence is a senescence inhibitor or a senescence-associated secretory phenotype (SASP) inhibitor.

[0269] In some embodiments, the senescence inhibitor is a senescence suppressor.

[0270] In some embodiments, the senescence inhibitor is a senescence-associated secretory phenotype (SASP) inhibitor. Preferably, the SASP inhibitor is an IL-1 inhibitor as described herein. Preferably, the SASP inhibitor is an NF-κB inhibitor as described herein.

[0271] Preferably, multiple senescence inhibitors can be used in combination. Preferably, a senescence inhibitor and a SASP inhibitor can be used in combination. Preferably, multiple senescence inhibitors and / or multiple SASP inhibitors can be used in combination. Thus, advantageously, inhibition of the SASP phenotype and slowing the rate at which senescent cells form can be achieved.

[0272] Thus, in some embodiments, the methods and uses of the present invention include the use of multiple aging inhibitors. In one embodiment, one or more aging inhibitors are combined with one or more SASP inhibitors. Preferably, the multiple aging inhibitors are different from each other. In other words, when multiple aging inhibitors are used, each inhibitor has a different target. Thus, the use of multiple inhibitors may provide additive or synergistic effects.

[0273] In some embodiments, the methods and uses of the present invention include the use of a senescence inhibitor (e.g., a MAPK / ERK signaling pathway inhibitor, e.g., a MAPK inhibitor), an IL-1 inhibitor, and / or an NF-κB inhibitor. Preferably, a senescence inhibitor (e.g., a MAPK / ERK signaling pathway inhibitor, e.g., a MAPK inhibitor) is used. Preferably, an IL-1 inhibitor is used. Preferably, an NF-κB inhibitor is used. Preferably, a senescence inhibitor (e.g., a MAPK / ERK signaling pathway inhibitor, e.g., a MAPK inhibitor) is used in combination with an IL-1 inhibitor. Preferably, a senescence inhibitor (e.g., a MAPK / ERK signaling pathway inhibitor, e.g., a MAPK inhibitor) is used in combination with an NF-κB inhibitor. Preferably, an IL-1 inhibitor is used in combination with an NF-κB inhibitor. Preferably, a senescence inhibitor (e.g., a MAPK / ERK signaling pathway inhibitor, e.g., a MAPK inhibitor), an IL-1 inhibitor, and an NF-κB inhibitor are used in combination.

[0274] The term "combination" or the terms "in combination with," "in combination with," or "combined preparation," as used herein, can refer to the simultaneous, sequential, or separate administration of two or more entities together.

[0275] In one embodiment, the senescence-suppressing agent (eg, a MAPK / ERK signaling pathway inhibitor, eg, a MAPK inhibitor), an IL-1 inhibitor, and / or an NF-κB inhibitor are administered simultaneously, sequentially, or separately.

[0276] In one embodiment, the senescence inhibitor (eg, a MAPK / ERK signaling pathway inhibitor, eg, a MAPK inhibitor), an IL-1 inhibitor, and / or an NF-κB inhibitor are administered simultaneously.

[0277] In one embodiment, the senescence-suppressing agent (eg, a MAPK / ERK signaling pathway inhibitor, eg, a MAPK inhibitor), an IL-1 inhibitor, and / or an NF-κB inhibitor are administered sequentially.

[0278] In one embodiment, the senescence-suppressing agent (eg, a MAPK / ERK signaling pathway inhibitor, eg, a MAPK inhibitor), an IL-1 inhibitor, and / or an NF-κB inhibitor are administered separately.

[0279] The term "concurrently" as used herein means that the entities are administered concurrently, i.e., at the same time.

[0280] The term "sequentially" as used herein means that the entities are administered one after the other.

[0281] The term "separately" as used herein means that the entities are administered independently of each other, but at a time interval that allows the entities to act additively, preferably synergistically. Thus, "separately" administration can allow for one entity to be administered, for example, within 1 minute, 5 minutes, or 10 minutes after the other entity.

[0282] In some embodiments, inhibition of senescence (e.g., MAPK / ERK signaling pathway inhibition, e.g., MAPK inhibition), IL-1 inhibition, and / or NF-κB inhibition in hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells is transient.

[0283] In one embodiment, the senescence inhibitor (e.g., MAPK / ERK signaling pathway inhibitor, e.g., MAPK inhibitor), IL-1 inhibitor and / or NF-κB inhibitor is a transient inhibitor (e.g., having an inhibitory effect lasting less than about 1, 2, 3, 4, 5, 6, 7 or 14 days), e.g., a reversible inhibitor. Suitably, the senescence inhibitor (e.g., MAPK / ERK signaling pathway inhibitor, e.g., MAPK inhibitor) is a transient inhibitor. Suitably, the IL-1 inhibitor is a transient inhibitor. Suitably, the NF-κB inhibitor is a transient inhibitor. Preferably, the cells are exposed to the inhibitor for about 1 to 48 or 1 to 24 hours, preferably 1 to 24 hours. The cells may be exposed to the inhibitor, for example, simultaneously with the viral vector or before the viral vector.

[0284] In some embodiments, hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are exposed to one or more senescence inhibitors before, concomitantly with and / or after the gene editing mechanism is introduced into the cells, preferably simultaneously with the gene editing mechanism being introduced into the cells.

[0285] In some embodiments, inhibition of senescence (e.g., MAPK inhibition), inhibition of IL-1 and / or NF-κB occurs upon gene editing of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells.

[0286] In some preferred embodiments, hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are exposed to a senescence inhibitor (particularly a senescence suppressor, e.g., a MAPK inhibitor) before the gene editing machinery is introduced into the cells.

[0287] In some preferred embodiments, MAPK / ERK signaling inhibition (e.g., MAPK inhibition) occurs before and / or during gene editing of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells.

[0288] In some embodiments, hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are exposed to one or more senescence inhibitors before, concomitantly and / or after the cells are transduced with one or more viral vectors, preferably simultaneously with the cells being transduced with one or more viral vectors.

[0289] In some embodiments, inhibition of senescence (e.g., MAPK inhibition), inhibition of IL-1 and / or NF-κB occurs during transduction of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more viral vectors.

[0290] In some preferred embodiments, hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are exposed to a senescence inhibitor (particularly a senescence suppressing agent, e.g., a MAPK inhibitor) prior to transducing the cells with one or more viral vectors.

[0291] In some preferred embodiments, MAPK / ERK signaling inhibition (e.g., MAPK inhibition) occurs prior to and / or during transduction of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more viral vectors.

[0292] In some embodiments, the MAPK inhibitor, IL-1 inhibitor and / or NF-κB inhibitor is added to the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells at a concentration of about 0.5 to 200 μM or about 0.1 to 200 ng / μL.

[0293] In one embodiment, the MAPK inhibitor, IL-1 inhibitor and / or NF-κB inhibitor is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 0.5 to 200, 0.5 to 150, 0.5 to 100, 0.5 to 50, 0.5 to 40, 0.5 to 30, 0.5 to 20, or 0.5 to 15 μM, preferably about 0.5 to 30 μM, more preferably about 0.5 to 15 μM. In one embodiment, the MAPK inhibitor, IL-1 inhibitor and / or NF-κB inhibitor is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 1-200, 1-150, 1-100, 1-50, 1-40, 1-30, 1-20 or 1-15 μM, preferably about 1-30 μM, more preferably about 1-15 μM. In another embodiment, the inhibitor is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells at a concentration of about 5-200, 5-150, 5-100, 5-50, 5-40, 5-30, 5-20 or 5-15 μM, preferably about 5-30 μM.

[0294] In one embodiment, the MAPK inhibitor, IL-1 inhibitor and / or NF-κB inhibitor is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175 or 200 μM.

[0295] In one embodiment, the MAPK inhibitor, IL-1 inhibitor and / or NF-κB inhibitor is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 0.1 to 200, 0.1 to 150, 0.1 to 100, 0.1 to 75, 0.1 to 60, 0.1 to 50, 0.1 to 25, 0.1 to 20, 0.1 to 15, or 0.1 to 10 ng / μL, preferably about 0.1 to 60 ng / μL. In another embodiment, the inhibitor is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells at a concentration of about 5 to 200, 5 to 150, 5 to 100, 5 to 75, 5 to 60, 5 to 50, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 ng / μL, preferably about 5 to 60 ng / μL.

[0296] In one embodiment, the MAPK inhibitor, IL-1 inhibitor and / or NF-κB inhibitor is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, or 200 ng / μL, preferably about 50 ng / μL.

[0297] When a concentration of a combination of inhibitors is given herein, it is understood that the concentration refers to each individual inhibitor within the combination. For example, when it is stated that a MAPK inhibitor, an IL-1 inhibitor, and an NF-κB inhibitor are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells at a concentration of about 0.5-200 μM or about 0.1-200 ng / μL, this means that the MAPK inhibitor is added at a concentration of about 0.5-200 μM or about 0.1-200 ng / μL, the IL-1 inhibitor is added at a concentration of about 0.5-200 μM or about 0.1-200 ng / μL, and the NF-κB inhibitor is added at a concentration of about 0.5-200 μM or about 0.1-200 ng / μL.

[0298] In one embodiment, the one or more senescence inhibitors comprise or consist of a p38 inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor. Suitably, the one or more senescence inhibitors comprise or consist of SB203580 or a derivative thereof, anakinra and / or SC514 or a derivative thereof.

[0299] In one embodiment, the one or more senescence inhibitors comprise or consist of a JNK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor. Suitably, the one or more senescence inhibitors comprise or consist of SP600125 or a derivative thereof, anakinra and / or SC514 or a derivative thereof.

[0300] In one embodiment, the one or more senescence inhibitors comprise or consist of an ERK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor. Suitably, the one or more senescence inhibitors comprise or consist of FR180204 or a derivative thereof, anakinra and / or SC514 or a derivative thereof.

[0301] Candidate inhibitors can be analyzed for their ability to increase cell survival and / or engraftment using methods as disclosed herein. For example, candidate inhibitors can be analyzed for their ability to increase clonogenicity using methods as disclosed herein. Candidate inhibitors can also be analyzed for their ability to increase gene editing efficiency using methods as disclosed herein. Candidate inhibitors can be analyzed for their ability to reduce p21 levels as described herein. Candidate inhibitors can be analyzed for their ability to attenuate inflammatory programs as a result of gene editing as described herein.

[0302] Anti-aging agent The mammalian mitogen-activated protein kinase (MAPK) family of kinases includes three subfamilies: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38). In general, ERKs are activated by growth factors and mitogens, while JNK and p38 are activated by cell stress and inflammatory cytokines. "Classical" MAPKs are activated by phosphorylation events at their activation loops (generally, activation depends on two phosphorylation events), forming a three-tiered signaling pathway. Phosphorylation of the tandem MAPK activation loops is accomplished by members of the Ste7 protein kinase family, also known as MAP2 kinases (MAP2Ks). MAP2Ks are in turn activated by phosphorylation accomplished by several different upstream serine-threonine kinases called MAP3 kinases (MAP3Ks). Because most MAP2Ks show little activity toward substrates other than their cognate MAPKs, the classical MAPK pathway is multi-tiered but relatively linear. "Atypical" MAPKs do not have dual phosphorylation sites and form only a two-tiered pathway.

[0303] In some embodiments, the senescence inhibitor (e.g., senescence suppressor) is an inhibitor of the mitogen-activated protein kinase (MAPK) / extracellular signal-regulated kinase (ERK) signaling pathway. Inhibition of the MAPK / ERK signaling pathway can be determined using methods known in the art. Suitably, the inhibitor is a MAP3K inhibitor, a MAK2K inhibitor, a MAPK inhibitor, preferably, the inhibitor is a MKK7 inhibitor, a MKK4 inhibitor, a MKK3 / 6 inhibitor, a MEK1 / 2 inhibitor, a JNK inhibitor, a p38 inhibitor, or an ERK inhibitor.

[0304] In some preferred embodiments, the MAPK / ERK signaling inhibitor is a MAPK inhibitor.

[0305] The activity of MAPK can be assayed directly, for example, by assaying the enzymatic activity of MAPK in vitro.

[0306] The ability of a candidate drug to inhibit (e.g., reduce) the activity of a MAPK is determined by IC 50 The inhibitors of the present invention can be expressed as an IC value, which is the concentration of agent required to produce a 50% decrease in the activity of the kinase. Preferably, the inhibitors of the present invention have an IC value of less than 100 μM for inhibition of (e.g., MAPK), more preferably less than 10 μM, e.g., less than 1 μM, less than 100 nM, or less than 10 nM. 50 It has a value.

[0307] Several techniques for measuring kinase activity are known in the art. Preferably, kinase activity assays are performed on kinases (e.g., MAPKs) isolated from cells. Kinases may be expressed using recombinant techniques and are preferably purified. For example, kinase activity can be determined by monitoring the incorporation of radiolabeled phosphate from [γ-32P]-labeled ATP into a substrate. Such assay techniques are described, for example, in Hastie et al. (Hastie, CJ et al. (2006) Nat. Protocols 1: 968-971).

[0308] Preferably, the MAPK inhibitor is a p38 phosphorylation inhibitor, a JNK phosphorylation inhibitor or an ERK phosphorylation inhibitor, preferably a p38 phosphorylation inhibitor.

[0309] In some preferred embodiments, the MAPK inhibitor is a JNK inhibitor, a p38 inhibitor or an ERK inhibitor, preferably a p38 inhibitor.

[0310] In some embodiments, the MAPK inhibitor is FR180204, SP600125, SB203580, SB202190, LY2228820, BIRB796, TAT-TN13, SB203580 hydrochloride, AMG548, SB239063, CMPD-1, JX401, EO 1428, RWJ67657, SCIO 469 hydrochloride, VX745, TAK715, ML3403, AL8697, SB706504, DBM1285 dihydrochloride, PH797804, Org48762-0, TMCB, XMD8-92, Pluripotin, TCS ERK 11e, ERK5-IN-1, DEL22379, AX15836, TCS JNK 6o, SU3327, CEP1347, c-JUN peptide, AEG3482, TCS JNK 5a, BI78D3, IQ3, SR3576, CC401 dihydrochloride, or a variant or derivative thereof.

[0311] In some embodiments, the MAPK inhibitor is FR180204, SP600125, SB203580, SB202190, LY2228820, BIRB 796; SB203580 hydrochloride, SCIO 469 hydrochloride, TMCB, XMD8-92, TCS JNK 6o, SU3327, CC401 dihydrochloride, or a variant or derivative thereof.

[0312] In some embodiments, the MAPK inhibitor is a JNK inhibitor.

[0313] In some embodiments, the JNK inhibitor is SP600125, TCS JNK 6o, SU3327, CEP1347, c-JUN peptide, AEG 3482, TCS JNK 5a, BI78D3, IQ3, SR3576, CC401 dihydrochloride, or a derivative thereof.

[0314] In some embodiments, the JNK inhibitor is SP600125, TCS JNK 6o, SU3327, CC401 dihydrochloride, or a derivative thereof.

[0315] In some preferred embodiments, the MAPK inhibitor is SP600125 or a derivative thereof.

[0316] SP600125 (C14H8N2O; anthra[1-9-cd]pyrazol-6(2H)-one) is a selective JNK inhibitor. SP600125 (CAS No. 129-56-6), also known as JNK inhibitor II, is a cell-permeable, potent, selective, ATP-competitive, and reversible inhibitor of JNK. SP600125 is active in vivo, competitively and reversibly inhibiting JNK1, 2, and 3 (IC 50 =40-90 nM) and has negligible activity against ERK2, p38β and a range of other enzymes. This inhibitor protects renal tubular epithelial cells from ischemia / reperfusion-induced apoptosis.

[0317] In one embodiment, SP600125 has the following structure: [ka]

[0318] TCS JNK 6o(C 18 H 20 N4O4 (N-(4-amino-5-cyano-6-ethoxy-2-pyridinyl)-2,5-dimethoxybenzeneacetamide) is an ATP-competitive JNK inhibitor (IC 50 (The EC values ​​are 2, 4, and 52 nM for JNK1, JNK2, and JNK3, respectively. TCS JNK 6o exhibits over 1000-fold selectivity over other kinases, including ERK2 and p38. TCS JNK 6o inhibits phosphorylation of c-Jun (EC 50 =920 nM), inhibiting collagen-induced platelet aggregation in vitro.

[0319] SU3327 (C5H3N5O2S3; 5-[(5-nitro-2-thiazolyl)thio]-1,3,4 thiadiazol-2-amine) is a selective inhibitor of JNK (IC 50= 0.7 μM). This inhibitor exhibits selectivity for p38 MAPK and Akt and inhibits the protein-protein interaction between JNK and JIP (IC 50 =239nM).

[0320] CC401 dihydrochloride (C 22 H 24 NO·2HCl;3-[3-[2-(1-piperidinyl)ethoxy]phenyl]-5-(1H-1,2,4-triazol-5-yl)-1H-indazole dihydrochloride) is a high affinity JNK inhibitor (Ki value is 25-50 nM). CC401 dihydrochloride inhibits JNK via competitive binding of the ATP binding site of activated phosphorylated JNK. This inhibitor shows >40-fold selectivity for JNK over p38, ERK, IKK2, protein kinase C, Lck and ZAP70. CC401 dihydrochloride is hepatoprotective and also inhibits HCMV replication.

[0321] Other JNK inhibitors include: CEP1347, a JNK signaling inhibitor; c-JUN peptide, a JNK / c-Jun interaction peptide inhibitor; AEG3482, a JNK signaling inhibitor; TCS JNK 5a, a selective inhibitor of JNK2 and JNK3; BI78D3, a selective competitive JNK inhibitor; IQ3, a selective JNK3 inhibitor; and SR3576a, a highly potent selective JNK3 inhibitor.

[0322] In some preferred embodiments, the MAPK inhibitor is a p38 inhibitor.

[0323] In some embodiments, the p38 inhibitor is SB203580, SB202190, LY2228820, BIRB796; TAT-TN13, SB203580 hydrochloride, AMG548, SB239063, CMPD-1, JX401, EO 1428, RWJ67657, SCIO 469 hydrochloride, VX745, TAK715, ML3403, AL8697, SB706504, DBM1285 dihydrochloride, PH797804, Org48762-0, or a variant or derivative thereof.

[0324] In some embodiments, the p38 inhibitor is SB203580, SB203580 hydrochloride, SB202190, LY2228820, BIRB796, SCIO469 hydrochloride, or a derivative thereof.

[0325] In some preferred embodiments, the MAPK inhibitor is SB203580, SB203580 hydrochloride, or a derivative thereof.

[0326] In some preferred embodiments, the MAPK inhibitor is SB203580 or a derivative thereof.

[0327] SB203580 (CAS No. 152121-47-6) is a highly specific, potent, cell-permeable, selective, reversible, and ATP-competitive inhibitor of p38 MAP kinase. SB203580 hydrochloride is water-soluble. SB203580(C 20 H 14 N3OF; 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]phenol), and SB203580 hydrochloride, are pyridinylimidazoles that suppress activation of MAPKAP kinase-2 and inhibit phosphorylation of heat shock protein (HSP) 27 in vivo in response to IL-1, cellular stress, and bacterial endotoxin. It does not inhibit JNK or p42 MAP kinases, making it useful for studying the physiological roles and targets of p38 MAPK and MAPKAP kinase-2. It has been shown to induce activation of the serine / threonine kinase Raf-1 and has been reported to inhibit cytokine production.

[0328] In one embodiment, SB203580 has the following structure: [ka]

[0329] SB202190(C 20 H 14 N 3OF; 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]phenol: SB202190 is a selective p38 MAP kinase inhibitor with IC50 values ​​against p38α and p38β2. 50 are 50 nM and 100 nM, respectively. SB202190 binds to the ATP pocket of active recombinant human p38 kinase with a Kd of 38 nM. SB202190 has anti-cancer activity and reversed memory impairment.

[0330] LY2228820(C 24 H 29 FN6·2CH3SO3H), also known as ralimetinib, is a trisubstituted imidazole derivative and a potent inhibitor of the α- and β-isoforms of p38 MAP kinase (MAPK) in vitro (IC 50 = 5.3 and 3.2 nM, respectively).

[0331] BIRB796(C 31 H 37 N5O3;N-[3-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazol-5-yl]-N'-[4-[2-(4-morpholinyl)ethoxy]-1-naphthalenyl]urea) (also known as dorampimod) is an orally active, highly potent p38 MAPK inhibitor. Dorampimod (BIRB 796) is commonly associated with inflammation due to its role in T-cell proliferation and cytokine production. Dorampimod (BIRB 796) blocks stress-induced phosphorylation of the scaffold protein SAP97, which has also been identified as a physiological substrate of SAPK3 / p38γ. Binding of dorampimod to p38 MAPK or JNK1 / 2 impairs their phosphorylation by the upstream kinases MKK6 or MKK4.

[0332] SCIO469 Hydrochloride (C 27 H 30ClFN4O3·HCl;6-chloro-5-[[(2R,5S)-4-[(4-fluorophenyl)methyl]-2,5-dimethyl-1-piperazinyl]carbonyl]-N,N,1-trimethyl-α-oxo-1H-indole-3-acetamide hydrochloride) (CAS No. 309913-83-5), also known as talmapimod, is an orally active, selective, ATP-competitive inhibitor of p38α with IC 50 The potency of talmapimod is 9 nM. Talmapimod exhibits approximately 10-fold selectivity for p38β and at least 2000-fold selectivity over a panel of 20 other kinases, including other MAPKs. Talmapimod specifically blocks cytokine-induced phosphorylation of p38 and inhibits CD34. + It reduces apoptosis of HSPCs and increases their colony-forming ability.

[0333] Other p38 inhibitors include: TAT-TN13, a selective p38 kinase inhibitor; AMG548, a potent and selective p38α inhibitor; SB239063, a potent and selective p38 MAPK inhibitor with oral activity; CMPD-1, a selective inhibitor of p38α-mediated MK2a phosphorylation and a tubulin polymerization inhibitor; JX401, a potent and reversible p38α inhibitor; EO 1428, a selective p38α and p38β2 inhibitor; RWJ67657, a potent and selective p38α and p38β inhibitor; VX745, a potent and selective p38α inhibitor; TAK715, a potent p38 MAPK inhibitor that also has anti-inflammatory properties; ML3403, a p38 inhibitor; AL8697, a potent and selective p38α inhibitor; SB706504, a p38 MAPK inhibitor; p38 DBM1285 dihydrochloride, a MAPK inhibitor that also has anti-inflammatory properties; PH797804, a potent and selective p38α / β inhibitor; and Org48762-0, an orally bioavailable selective p38α / β inhibitor.

[0334] In some embodiments, the MAPK inhibitor is an ERK inhibitor.

[0335] In some embodiments, the ERK inhibitor is FR180204, TMCB, XMD8-92, pluripotin, TCS ERK 11e, ERK5-IN-1, DEL22379, AX15836, or a variant or derivative thereof.

[0336] In some embodiments, the ERK inhibitor is FR180204, TMCB, XMD8-92, or a derivative thereof.

[0337] In some preferred embodiments, the MAPK inhibitor is FR180204 or a derivative thereof.

[0338] FR180204 (CAS No. 865362-74-9) is a cell-permeable, potent, ATP-competitive inhibitor of ERK1 and ERK2. 18 H 13 N7; 5-(2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine) is a selective ERK inhibitor (IC for ERK2 and ERK1). 50 The IC values ​​are 0.14 and 0.31 μM, respectively. FR180204 exhibits 30-fold selectivity for ERK over p38α (IC 50 = 10 μM); it has no activity against human recombinant MEK1, MKK4, IKKα, PKCα, Src, Syc and PDGFα at concentrations below 30 μM. FR180204 also inhibits TGFβ-induced AP-1 activation in Mv1Lu cells (IC 50 =3.1μM).

[0339] In some embodiments, FR180204 has the following structure: [ka]

[0340] TMCB(C 11H9Br4N3O2;2-(4,5,6,7-tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazol-1-yl)acetic acid) is a dual kinase inhibitor that inhibits both casein kinase 2 (CK2) and extracellular signal-regulated kinase 8 (ERK8) (IC 50 = 0.50 μM). TMCB exhibits selectivity for CK2 over protein kinases normally sensitive to CK2 inhibitors (Ki values ​​for CK2, PIM1, DYRK1a and HIPK2 are 0.25, 8.65, 11.90 and 15.25 μM, respectively).

[0341] XMD8-92(C 26 H 30 N6O3;2-[[2-ethoxy-4-(4-hydroxy-1-piperidinyl)phenyl]amino]-5,11-dihydro-5,11-dimethyl-6H-pyrimod[4,5-b][1,4]benzodizepin-6-one) is an ERK5 (BMK1) and BRD4 inhibitor (K d The K values ​​are 80 and 190 nM, respectively. XMD 8-92 also inhibits DCAMKL2, PLK4, and TNK1 (K d (Values ​​are 190, 600 and 890 nM). XMD8-92 blocks growth factor-induced activation of cellular BMK1 and reduces BMK1 activity in in vitro kinase assays. The inhibitor also reduces BMK1-dependent transactivated MEF2C activity. XMD8-92 inhibits the proliferation of various cancer cell lines and blocks tumor cell proliferation and tumor-associated angiogenesis.

[0342] Other ERK inhibitors include: pluripotin, a dual ERK1 / RasGAP inhibitor that maintains ESC self-renewal; TCS ERK 11e, a potent and selective ERK2 inhibitor; ERK5-IN-1, a potent and selective ERK5 inhibitor; DEL22379, an ERK dimerization inhibitor; and AX15836, a potent and selective ERK5 inhibitor.

[0343] In some embodiments, the MAPK inhibitor is SB203580, FR180204, SP600125 or a derivative thereof.

[0344] In some embodiments, the senescence inhibitor is not an IL-1 inhibitor.

[0345] In some embodiments, the senescence inhibitor is not an NF-κB inhibitor.

[0346] In some embodiments, the senescence inhibitor is not a p53 activation inhibitor. Suitably, the senescence inhibitor is not GSE56. Suitably, the senescence inhibitor is not GSE56 or a variant thereof.

[0347] In some embodiments, the senescence inhibitor is not an IL-1 inhibitor, an NF-κB inhibitor, or a p53 activation inhibitor.

[0348] In one embodiment, the MAPK inhibitor (e.g., SB203580, FR180204, SP600125, or a derivative thereof) is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 1 to 200, 1 to 150, 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 15 μM, preferably about 1 to 15 μM.

[0349] In one embodiment, a MAPK inhibitor (e.g., SB203580, FR180204, SP600125 or a derivative thereof) is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20 μM.

[0350] In another embodiment, the MAPK inhibitor (e.g., FR180204 or a derivative thereof) is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 12 μM.

[0351] In another embodiment, a MAPK inhibitor (e.g., SP600125 or a derivative thereof) is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 2 μM.

[0352] In another embodiment, a MAPK inhibitor (e.g., SB203580 or a derivative thereof) is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 4 μM.

[0353] In one embodiment, the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are contacted with a MAPK inhibitor for about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours, preferably about 24 hours or 48 hours, prior to transducing the cell population with a viral vector and / or prior to introducing a gene editing mechanism into the cells.

[0354] In one embodiment, the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are contacted with the MAPK inhibitor for about 15 minutes to about 4 hours; about 15 minutes to about 3 hours; or about 15 minutes to about 2 hours after transducing the cell population with one or more viral vectors and / or introducing a gene editing mechanism into the cells. In another embodiment, the cells are contacted with the inhibitor for about 1 to 4 hours; 1 to 3 hours; or 1 to 2 hours after transducing the cell population with a viral vector and / or introducing a gene editing mechanism into the cells (e.g., after electroporating the cells).

[0355] In one embodiment, the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are contacted with a MAPK inhibitor for about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours or 4 hours, preferably about 15 minutes, after transduction of the cell population with a viral vector and / or after introduction of a gene editing mechanism into the cells (e.g., after electroporation of the cells).

[0356] Suitably, the MAPK inhibitor may be active during gene editing.

[0357] Advantageously, the MAPK inhibitor may be active during transduction.

[0358] In one embodiment, the cells are contacted with the MAPK inhibitor for about 12 to 96 hours, e.g., 12 to 60 hours, 24 to 60 hours, 36 to 60 hours, or 42 to 54 hours, preferably about 42 to 54 hours, after introduction of the gene editing mechanism and / or viral vector into the cells. In one embodiment, the contacting is performed for about 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 hours, preferably about 48 hours or about 96 hours, after initiation of introduction and / or transduction into the cells.

[0359] In one embodiment, the cells are contacted with the MAPK inhibitor for about 12-60 hours, e.g., 24-60 hours, 36-60 hours, or 42-54 hours, preferably about 42-54 hours, after initiating culture of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., culturing is initiated after the cells are thawed from a frozen state). In one embodiment, the cells are contacted with the MAPK inhibitor for about 12, 18, 24, 30, 36, 42, 48, 54, or 60 hours, preferably about 48 hours, after initiating culture of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0360] In one embodiment, the cells are contacted with the MAPK inhibitor for about 12-60 hours, e.g., 24-60 hours, 36-60 hours, or 42-54 hours, preferably about 42-54 hours, after thawing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., stored frozen). In one embodiment, the cells are contacted with the MAPK inhibitor for about 12, 18, 24, 30, 36, 42, 48, 54, or 60 hours, preferably about 48 hours, after thawing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0361] In one embodiment, the cells are contacted with the MAPK inhibitor for about 12-60 hours, e.g., 24-60 hours, 36-60 hours, or 42-54 hours, preferably about 42-54 hours, prior to introducing the gene editing mechanism and / or viral vector into the cells. In one embodiment, the contacting occurs for about 12, 18, 24, 30, 36, 42, 48, 54, or 60 hours, preferably about 48 hours, prior to initiating introduction and / or transduction into the cells.

[0362] In one embodiment, the cells are contacted with the MAPK inhibitor for about 12-96 hours, e.g., 12-60 hours, 24-60 hours, 36-60 hours, or 42-54 hours, preferably about 42-54 hours, after the gene editing mechanism and / or viral vector is introduced into the cells. In one embodiment, the contacting occurs for about 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 hours, preferably about 48 hours or about 96 hours, after the introduction and / or transduction step is initiated.

[0363] In one embodiment, the cells are contacted with the MAPK inhibitor for about 12-96 hours, e.g., 12-72 hours, 12-60 hours, 24-60 hours, 36-60 hours, or 42-54 hours, preferably about 72-96 hours, after initiating culture of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., initiating culture after the cells are thawed from a frozen state). In one embodiment, the cells are contacted with the MAPK inhibitor for about 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102 hours, preferably about 72 hours, after initiating culture of a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0364] In one embodiment, the cells are contacted with the MAPK inhibitor for about 12 to 96 hours, e.g., 12 to 60 hours, 24 to 60 hours, 36 to 60 hours, or 42 to 54 hours, preferably about 72 to 96 hours, after thawing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., stored frozen). In one embodiment, the cells are contacted with the MAPK inhibitor for about 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, or 102 hours, preferably about 72 hours, after thawing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0365] IL-1 inhibitors In some embodiments, the IL-1 inhibitor is an anti-IL-1α antibody, an anti-IL-1β antibody, an IL-1 antagonist, an IL-1 receptor antagonist, an IL-1α converting enzyme inhibitor, an IL-1β converting enzyme inhibitor, or a soluble decoy IL-1 receptor.

[0366] In some embodiments, the IL-1 inhibitor is IL-1Ra, anakinra, canakinumab, rilonacept, gevokizumab or a variant thereof; or LY2189102, MABpl, MEDI-8968, CYT013, slL-1 Rl, slL-1 Rll, EBI-005, CMPX-1023, VX-76, or a variant or derivative thereof.

[0367] In some embodiments, the IL-1 inhibitor is IL-1Ra, anakinra, canakinumab, rilonacept, gevokizumab, or a variant thereof.

[0368] In some embodiments, the IL-1 inhibitor is anakinra, canakinumab, rilonacept, gevokizumab, or variants thereof.

[0369] In one embodiment, the IL-1 inhibitor is canakinumab or a variant thereof.

[0370] In one embodiment, the IL-1 inhibitor is rilonacept or a variant thereof.

[0371] In one embodiment, the IL-1 inhibitor is gevokizumab or a variant thereof.

[0372] In a preferred embodiment, the IL-1 inhibitor is anakinra or a variant thereof.

[0373] An IL-1 inhibitor can be an antagonist of IL-1α or IL-1β. An agonist is a chemical that binds to a receptor and activates the receptor to produce a biological response, such as interleukin-1. In contrast, an antagonist blocks the action of an agonist.

[0374] The interleukin-1 (IL-1) family is a group of 11 cytokines that induce a complex network of proinflammatory cytokines and regulate and induce inflammatory responses (through the expression of integrins on leukocytes and endothelial cells). IL-1α and IL-1β are the best-studied members. IL-1α and IL-1β contain a β-trefoil fold and bind to the IL-1 receptor (IL-1R). Binding of IL-1α and IL-1β to the IL-1 receptor (IL-1R) promotes recruitment of the IL-1 receptor accessory protein (IL-1RAcP) and further signaling through the MyD88 adaptor.

[0375] IL-1α and IL-1β have a natural antagonist: the IL-1 receptor antagonist (IL-1Ra). IL-1Ra also contains a β-trefoil fold and binds to the IL-1R. Binding of IL-1Ra to the IL-1R prevents the recruitment of IL-1RAcP. Thus, IL-1Ra regulates (and inhibits) the proinflammatory activity of IL-1α and IL-1β by competing with them for binding sites on the receptor. IL-1Ra functions as a competitive inhibitor of the IL-1 receptor in vivo and in vitro. IL-1Ra counteracts the effects of both IL-1α and IL-1β. When bound by IL-1Ra, the IL-1 receptor does not transmit signals to the cell.

[0376] In one embodiment, the IL-1 inhibitor is an interleukin-1 receptor antagonist (IL-1Ra).

[0377] Anakinra (C 759 H 1186 N 208 O 232 S 10 A recombinant and slightly modified form of human interleukin-1 receptor antagonist (IL-1Ra), known as anakinra, is commercially available under the product Kineret® and sold by Swedish Orphan Biovitrum. Anakinra can be produced by recombinant DNA technology in Escherichia coli cells. Anakinra differs from the sequence of human IL-1Ra by the addition of an additional methionine at the N-terminus; it also differs from the human protein in that it is not glycosylated, for example, when produced in E. coli. Anakinra is a biopharmaceutical used to treat rheumatoid arthritis, cryopyrin-associated periodic syndromes, familial Mediterranean fever, and Still's disease. Anakinra is administered by subcutaneous injection.

[0378] An exemplary sequence of anakinra is shown in SEQ ID NO:3: TIFF2024544746000004.tif16161

[0379] In one embodiment, the IL-1 inhibitor is anakinra or an amino acid sequence having at least 80% (preferably at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:3, and optionally the inhibitor consists of the amino acid sequence of SEQ ID NO:3.

[0380] Canakinumab (C 6452 H 9958 N 1722 O 2010 S 42 ) binds to human IL-1β and neutralizes its inflammatory activity by blocking interaction with the IL-1 receptor, but has no cross-reactivity with other members of the IL-1 family, including IL-1α or IL-1Ra. Canakinumab is commercially available from Novartis under the trademark Ilaris®. Canakinumab, also known as ACZ885, is a recombinant human monoclonal antibody that targets human IL-1β. Canakinumab belongs to the IgG1 / κ isotype subclass. Canakinumab is a drug for the treatment of active Still's disease, including juvenile idiopathic arthritis (SJIA) and adult-onset Still's disease (AOSD). Canakinumab contains two heavy chains and two light chains. Both heavy chains of canakinumab contain oligosaccharide chains linked to the protein backbone at asparagine 298 (Asn298).

[0381] An exemplary sequence of the heavy chain of canakinumab is shown in SEQ ID NO:4. TIFF2024544746000005.tif37163

[0382] An exemplary sequence of the light chain of canakinumab is shown in SEQ ID NO:5. TIFF2024544746000006.tif22161

[0383] In one embodiment, the IL-1 inhibitor is canakinumab or an antibody comprising two heavy chains and two light chains, each heavy chain having at least 80% (preferably at least 85%, at least 90%, at least 95%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 4, and optionally consisting of SEQ ID NO: 4; and each light chain having at least 80% (preferably at least 8, at least 90%, at least 95%, or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 5, and optionally consisting of SEQ ID NO: 5.

[0384] Gevokizumab (C 6442 H 9962 N 1710 O 2010 S 52 IL-1β is a monoclonal antibody that binds to the proinflammatory cytokine IL-1β and downregulates cell signaling events that lead to inflammation. IL-1β has been implicated in cardiovascular pathology, lung cancer, and autoinflammatory diseases. Gevokizumab is an investigational monoclonal antibody developed by XOMA Corporation that has allosteric modulatory properties.

[0385] Rilonacept (C 9030 H 13932 N 2400 O 2670 S 74 Rilonacept is a dimeric fusion protein consisting of the ligand-binding domains of the human interleukin-1 receptor component (IL-1R1) and the extracellular portion of the IL-1 receptor accessory protein (IL-1RAcP) linked in tandem to the fragment-crystallizable portion of human IgG1 (Fc region), which binds and neutralizes IL-1. Rilonacept is commercially available as ARCALYST® from Regeneron. Rilonacept is a soluble decoy receptor.

[0386] An exemplary sequence for rilonacept is shown in SEQ ID NO:6. TIFF2024544746000007.tif26164TIFF2024544746000008.tif47161

[0387] In one embodiment, the IL-1 inhibitor is an amino acid sequence having at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 99%) sequence identity to rilonacept or the amino acid sequence of SEQ ID NO:6, and optionally the inhibitor consists of the amino acid sequence of SEQ ID NO:6.

[0388] In one embodiment, an IL-1 inhibitor (e.g., anakinra) is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 0.1 to 200, 0.1 to 150, 0.1 to 100, 0.1 to 75, 0.1 to 60, 0.1 to 50, 0.1 to 25, 0.1 to 20, 0.1 to 15, or 0.1 to 10 ng / μL, preferably about 0.1 to 60 ng / μL. In another embodiment, an IL-1 inhibitor (e.g., anakinra) is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells at a concentration of about 5-200, 5-150, 5-100, 5-75, 5-60, 5-50, 5-25, 5-20, 5-15, or 5-10 ng / μL, preferably about 5-60 ng / μL.

[0389] In one embodiment, an IL-1 inhibitor (e.g., anakinra) is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., during in vitro or ex vivo culture) at a concentration of about 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or 100 ng / μL, preferably about 50 ng / μL.

[0390] NF-κB inhibitors Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a protein complex that controls DNA transcription, cytokine production and cell survival. NF-κB is found in almost all animal cell types and is involved in cellular responses to a wide range of stimuli, including stress, cytokines, free radicals, TNFα, IL-1β and bacterial or viral antigens.

[0391] In unstimulated cells, NF-κB dimers are sequestered in an inactive state in the cytoplasm by a family of inhibitors called inhibitors of κB (IκBs), which contain multiple copies of ankyrin repeats. IκB proteins mask the nuclear localization signal (NLS) of NF-κB proteins via the ankyrin repeat domain. The IκB family consists of IκBα, IκBβ, IκBε, and Bcl-3.

[0392] NF-κB activation is initiated by signal-induced degradation of IκB proteins. This occurs primarily through the activation of a kinase called IκB kinase (IKK). IKK contains a heterodimer of catalytic IKKα and IKKβ subunits and a regulatory protein called NF-κB essential modulator (NEMO) or IKKγ. Upon activation by a signal (usually an extracellular signal), IKK phosphorylates two serine residues located within the IκB regulatory domain. The phosphorylated IκB proteins are ubiquitinated, leading to their degradation by the proteasome. Degradation of IκB releases the NF-κB complex, which can translocate to the nucleus and act as a transcription factor for many NF-κB target genes.

[0393] The NF-κB inhibitor may act at any stage of NF-κB activation and / or at any point in the NF-κB signaling pathway. Suitably, the NF-κB inhibitor is an IL-1 inhibitor.

[0394] In one embodiment, the NF-κB inhibitor is not an IL-1 inhibitor.

[0395] In some embodiments, the NF-κB inhibitor is an IL-1 inhibitor, an IL-1 receptor inhibitor, a TLR4 inhibitor, a TAK1 inhibitor, an Akt inhibitor, an IKK inhibitor, an IκB phosphorylation inhibitor, an IκB degradation inhibitor, a proteasome inhibitor, an IκBα upregulation inhibitor, an NF-κB nuclear translocation inhibitor, an NF-κB expression inhibitor, an NF-κB DNA binding inhibitor, or an NF-κB transactivation inhibitor.

[0396] Kinase activity can be directly assayed, for example, by assaying the enzymatic activity of the kinase in vitro as described herein. The ability of a candidate agent to inhibit (e.g., reduce) the activity of a kinase is determined by IC 50 The inhibitors of the present invention can be expressed as an IC value, which is the concentration of agent required to produce a 50% decrease in the activity of the kinase. Preferably, the inhibitors of the present invention have an IC value of less than 100 μM for inhibition of (e.g., MAPK), more preferably less than 10 μM, e.g., less than 1 μM, less than 100 nM, or less than 10 nM. 50 It has a value.

[0397] In some embodiments, the NF-κB inhibitor is SC514 or a derivative thereof; IL-1Ra, anakinra, canakinumab, rilonacept, gevokizumab or a variant thereof; or LY2189102, MABpl, MEDI-8968, CYT013, slL-1 Rl, slL-1 Rll, EBI-005, CMPX-1023, VX-76 or a derivative thereof; or metformin, apigenin, kaempferol, BAY11-7082, simvastatin or a derivative thereof; or siRNA, shRNA, miRNA or antisense DNA / RNA.

[0398] In some embodiments, the NF-κB inhibitor is SC514 or a derivative thereof; anakinra, canakinumab, rilonacept, gevokizumab or a variant thereof; or metformin, apigenin, kaempferol, BAY 11-7082, or a derivative thereof.

[0399] In some embodiments, the NF-κB inhibitor is SC514 or a derivative thereof; IL-1Ra, anakinra, canakinumab, rilonacept, gevokizumab or a variant thereof; or siRNA, shRNA, miRNA or antisense DNA / RNA.

[0400] In some embodiments, the NF-κB inhibitor is an IKK inhibitor. In a preferred embodiment, the NF-κB inhibitor is SC514 or a derivative thereof.

[0401] SC514 (C9H8N2OS2) (CAS No. 354812-17-2) is a selective and reversible inhibitor of IKKβ (IKK-2) that exhibits greater than 10-fold selectivity over 28 other kinases, including JNK, p38, MK2, and ERK (IC 50 = 3-12 μM). This compound attenuates NF-κB-induced IL-6, IL-8, and Cox-2 gene expression (IC 50 = 20, 20, and 8 μM, respectively). Inappropriate activation of NF-κB has been linked to cancer, immune and inflammatory diseases, and viral infections.

[0402] In some embodiments, SC514 has the following structure: [ka]

[0403] Metformin (C4H 11 N5) is an IKK and / or NF-κB inhibitor. Metformin is approved for the treatment of type 2 diabetes.

[0404] Apigenin (C 15 H10 O5) is an inhibitor of the NF-κB p65 subunit and IκB. Apigenin is a natural flavonoid.

[0405] Kaempferol (C 15 H 10 O6) is an inhibitor of the NF-κB p65 subunit and IκB. Kaempferol is a natural flavonoid.

[0406] BAY11-7082(C 10 H9NO2S) is an inhibitor of NF-κB p65 subunit and IκB. BAY11-7082 has been used in preclinical models of aging in vitro. Bay11-7082 acts as a selective inhibitor of the nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway. In addition to inhibiting nuclear factor-κB (NF-κB), BAY11-7082 also induces apoptosis in anucleated erythrocytes, human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines, and primary adult T-cell leukemia cells. Bay11-7082 is an inhibitor of cytokine-induced IκB-α phosphorylation.

[0407] In one embodiment, the NF-κB inhibitor (e.g., SC514 or a derivative thereof) is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 1-200, 1-150, 1-100, 1-50, 1-40, 1-30, 1-20, or 1-15 μM, preferably about 1-30 μM. In another embodiment, the NF-κB inhibitor (e.g., SC514 or a derivative thereof) is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells at a concentration of about 5-200, 5-150, 5-100, 5-50, 5-40, 5-30, 5-20, or 5-15 μM, preferably about 5-30 μM.

[0408] In one embodiment, an NF-κB inhibitor (e.g., SC514 or a derivative thereof) is added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells (e.g., in in vitro or ex vivo culture) at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 μM, preferably 25 μM.

[0409] Agents that promote homology-dependent DNA repair The inventors have previously determined that improving HDR efficiency in HSCs increases cell survival and engraftment of gene-edited HSCs (see WO2020002380, which is incorporated by reference in its entirety). Suitable agents that promote HDR for use according to the present invention are described in WO2020002380, which is incorporated by reference in its entirety.

[0410] Gene editing in primary cells and HSPCs in particular can be hampered by gene transfer efficiency and limited HDR, likely due to low expression levels of HDR machinery and high activity of the NHEJ pathway.

[0411] As used herein, an "agent that promotes homology-dependent DNA repair" refers to an agent that enhances and / or improves HDR efficiency compared to the level of HDR in a cell that is not treated with the agent.

[0412] Suitably, the HDR may be increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Suitably, the HDR may be increased by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0 times or more.

[0413] HDR efficiency may be measured using any method in the art. Advantageously, HDR efficiency may be determined by FACS measurement of the presence of a marker in the donor template vector introduced into cells by HDR-mediated integration at the target locus. For example, the AAV6 donor template may contain a PGK.GFP reporter cassette. The increase in HDR efficiency may be measured by the presence of a marker in the donor template vector introduced into cells by HDR-mediated integration at the target locus. For example, the AAV6 donor template may contain a PGK.GFP reporter cassette. + This can be achieved by transient p53 inhibition, as measured by determining the percentage of cells

[0414] Targeted integration ("on-target" HDR) can be determined by digital PCR using primers and probes designed on the junction of the vector sequence and the target locus, as well as on a control sequence (human TTC5 gene) used for normalization.

[0415] The percentage of insertions and deletions (indels) introduced by the non-homologous end joining (NHEJ) repair pathway on nuclease target sites can be measured by mismatch-sensitive endonuclease assay (PCR-based amplification of the target locus followed by digestion with T7 endonuclease I; digested DNA fragments were separated and quantified by capillary electrophoresis on a LabChip GX Touch HT, Perkin Elmer). The level of mutations induced by NHEJ is used as a surrogate measurement to score nuclease activity.

[0416] In some embodiments, one or more senescence inhibitors are combined with an agent that promotes homology-dependent DNA repair (preferably, a p53 activation inhibitor). Preferably, the p53 activation inhibitor is GSE56 or a mutant thereof.

[0417] In some embodiments, the MAPK inhibitor, IL-1 inhibitor and / or NF-κB inhibitor is combined with an agent that promotes homology-dependent DNA repair (preferably, a p53 activation inhibitor).Preferably, the p53 activation inhibitor is GSE56 or a mutant thereof.

[0418] In some embodiments, the MAPK inhibitor is combined with an agent that promotes homology-dependent DNA repair (preferably, a p53 activation inhibitor). Preferably, the p53 activation inhibitor is GSE56 or a mutant thereof.

[0419] In some embodiments, the IL-1 inhibitor is combined with an agent that promotes homology-dependent DNA repair, preferably a p53 activation inhibitor. Preferably, the p53 activation inhibitor is GSE56 or a mutant thereof.

[0420] In some preferred embodiments, the NF-κB inhibitor is combined with an agent that promotes homology-dependent DNA repair (preferably, a p53 activation inhibitor). Preferably, the p53 activation inhibitor is GSE56 or a mutant thereof.

[0421] Preferably, the agent has low cytotoxicity.

[0422] Preferably, the agent does not significantly alter the composition of the gene-edited cells.

[0423] Preferably, the agent does not significantly induce differentiation of the gene-edited hematopoietic stem and / or progenitor cells, and thus the gene-edited hematopoietic stem and / or progenitor cells and / or T cells according to the invention retain their long-term repopulation potential.

[0424] Suitably, the composition or differentiation of the gene edited cells is altered by less than 5%, less than 4%, less than 3%, less than 2% or less than 1% compared to untreated controls.

[0425] p53 activation inhibitors In a preferred embodiment, the agent that promotes HDR is a p53 activation inhibitor.

[0426] Thus, in one aspect, the invention provides the use of (i) one or more senescence inhibitors and (ii) a p53 activation inhibitor to increase survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0427] In a further aspect, the present invention provides the use of (i) one or more senescence inhibitors and (ii) a p53 activation inhibitor to increase gene editing efficiency of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

[0428] In a further aspect, the present invention provides a combination comprising (i) one or more senescence inhibitors and (ii) a p53 activation inhibitor for use in hematopoietic cell gene therapy, hematopoietic stem cell gene therapy, hematopoietic progenitor cell gene therapy and / or T cell gene therapy.

[0429] In a further aspect, the present invention provides a combination comprising (i) one or more senescence inhibitors and (ii) a p53 activation inhibitor for use in increasing survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

[0430] In some embodiments, the p53 activation inhibitor is a p53 phosphorylation inhibitor, more preferably a p53 serine 15 phosphorylation inhibitor.

[0431] In some embodiments, the p53 activation inhibitor is a p53 dominant negative peptide, an ataxia telangiectasia mutated (ATM) kinase inhibitor or an ataxia telangiectasia and Rad3-related protein (ATR) inhibitor.

[0432] In some embodiments, the p53 activation inhibitor is pifithrin-α or a derivative thereof; KU-55933 or a derivative thereof; GSE56 or a mutant thereof; KU-60019, BEZ235, wortmannin, CP-466722, torin 2, CGK 733, KU-559403, AZD6738 or a derivative thereof; or siRNA, shRNA, miRNA or antisense DNA / RNA.

[0433] p53 dominant negative peptide In a preferred embodiment, the inhibitor of p53 activation is a mutant p53 peptide.

[0434] In a preferred embodiment, the inhibitor of P53 activation is a dominant negative peptide (eg, a dominant negative p53 peptide).

[0435] Advantageously, the dominant negative peptide may comprise a mutation in the homo-oligomerisation domain. Advantageously, the dominant negative peptide comprising a mutation in the homo-oligomerisation domain may dimerise with wild type p53 and prevent it from activating transcription.

[0436] In a preferred embodiment, the dominant negative peptide is GSE56 or a variant thereof.

[0437] In one embodiment, the nucleotide sequence for the mRNA translation of GSE56 is shown in SEQ ID NO:8. TIFF2024544746000010.tif27162

[0438] In one embodiment, the amino acid sequence of GSE56 is set forth in SEQ ID NO:9. TIFF2024544746000011.tif12162

[0439] Preferably, the inhibitor of p53 activation may be a nucleotide sequence encoding GSE56. Preferably, the inhibitor of p53 activation may be an amino acid sequence encoding GSE56. Preferably, the inhibitor of p53 activation may be GSE56 mRNA.

[0440] Kinase inhibitors In some embodiments, the agent that promotes HDR is an ataxia mutated (ATM) kinase inhibitor or an ataxia telangiectasia and Rad3 related protein (ATR) inhibitor.

[0441] The activity of ATM kinase and ATR can be assayed directly, for example, by assaying the enzymatic activity of ATM kinase or ATR in vitro.

[0442] The ability of a candidate agent to inhibit (e.g., reduce activity of) ATM kinase or ATR is determined by IC 50 The inhibitors of the present invention can be expressed in terms of an IC value, which is the concentration of drug required to produce a 50% decrease in kinase activity. Preferably, the inhibitors of the present invention have an IC value for inhibition (e.g., of ATM kinase or ATR) of less than 100 μM. 50 values, but more preferably less than 10 μM, e.g., less than 1 μM, less than 100 nM, or less than 10 nM (e.g., IC 50 The value is approximately 13 nM for ATM kinase).

[0443] Numerous techniques are known in the art for measuring kinase activity. Preferably, kinase activity assays are performed on kinases (e.g., ATM kinase or ATR) isolated from cells. The kinases may be recombinantly expressed and are preferably purified. For example, kinase activity can be measured by measuring the [γ- 32The activity of the ATP-activated ATP complex can be measured by monitoring the incorporation of radiolabeled phosphate from [P]-labeled ATP into the substrate. Such assay techniques are described, for example, in Hastie et al. (Hastie, CJ et al. (2006) Nat. Protocols 1: 968-971).

[0444] Preferably, the inhibitor has low toxicity to mammals, such as humans, and more particularly, low toxicity to hematopoietic stem and / or progenitor cells.

[0445] Candidate inhibitors can be further analyzed for their ability to increase cell survival and / or engraftment using the methods described herein.

[0446] Preferably, the inhibitor is a transient inhibitor (eg, having an inhibitory effect lasting less than about 1, 2, 3, 4, 5, 6, 7, or 14 days).

[0447] Preferably, the inhibitor is a pharmacological inhibitor.

[0448] In a preferred embodiment, the inhibitor is KU-55933 or a derivative thereof.

[0449] KU-55933 (CAS No. 587871-26-9) is a selective, competitive ATM kinase inhibitor and has the following structure: [ka]

[0450] Solutions of KU-55933 for use in the present invention can be prepared by conventional methods known in the art, for example, KU-55933 is known to be soluble in DMSO and ethanol.

[0451] The concentration at which KU-55933 or a derivative thereof is added to the hematopoietic stem and / or progenitor cell population can be adapted for different vector systems to optimize cell survival (e.g., during in vitro or ex vivo culture) and / or engraftment.

[0452] The present invention encompasses the use of KU-55933 and derivatives of KU-55933. KU-55933 derivatives for use in the present invention are those that increase the survival (e.g., in in vitro or ex vivo culture) and / or engraftment of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells, particularly cells transduced by viral vectors.

[0453] The KU-55933 derivatives of the present invention can be developed, for example, for increased solubility, increased stability, and / or reduced toxicity.

[0454] The KU-55933 derivatives of the present invention preferably have low toxicity to mammals, particularly humans.The KU-55933 derivatives of the present invention preferably have low toxicity to hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells and / or T cells.

[0455] Suitable KU-55933 derivatives can be identified using methods known in the art for measuring cell survival and / or engraftment in culture. Examples of such methods are described above. The method used is preferably one that is suitable for automation and / or high-throughput screening of potential KU-55933 derivatives. Potential KU-55933 derivatives may be part of a library of KU-55933 derivatives.

[0456] Further kinase inhibitors that can be used in the present invention include:

[0457] KU-60019, an improved analog of KU-55933, has an IC of 6.3 nM for ATM kinase in a cell-free assay. 50 KU-60019 has the following structure: [ka]

[0458] BEZ235 (NVP-BEZ235, Dactolisib), a dual ATP-competitive PI3K and mTOR inhibitor against p110α / γ / δ / β and mTOR (p70S6K), has an IC of approximately 21 nM in 3T3 TopBP1-ER cells. 50 BEZ235 has the following structure: [ka]

[0459] Wortmannin, which has the following structure: [ka]

[0460] CP-466722, which is a potent and reversible ATM kinase inhibitor but does not affect ATR. CP-466722 has the following structure: [ka]

[0461] Torin2, which has ECs of 28 nM and 35 nM for ATM kinase and ATR, respectively. 50 Torin2 has the following structure: [ka]

[0462] CGK733 (CAS No. 905973-89-9), a potent and selective inhibitor of ATM kinase and ATR, has been shown to be effective in treating 50 The value is approximately 200 nM. CGK733 has the following structure: [ka]

[0463] KU-559403 (Weber et al. (2015) Pharmacology & Therapeutics 149: 124-138). KU-559403 has the following structure: [ka]

[0464] AZD6738, which has the following structure: [ka]

[0465] Derivatives of the above inhibitors having the properties as described for the KU-55933 derivatives can also be used in the present invention and can be identified by methods similar to those described for the KU-55933 derivatives.

[0466] In a preferred embodiment, the p53 inhibitor can be pifithrin-α, cyclic pifithrin-α, and pifithrin-α p-nitro or a derivative thereof. Pifithrin-α has the following structure: [ka]

[0467] siRNA, shRNA, miRNA and antisense DNA / RNA Inhibition (e.g., inhibition of kinases) can be achieved using post-transcriptional gene silencing (PTGS). Post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA) is a conserved cellular defense mechanism for controlling the expression of foreign genes. Random integration of elements such as transposons and viruses is thought to cause the expression of dsRNA that activates sequence-specific degradation of homologous single-stranded mRNA or viral genomic RNA. The silencing effect is known as RNA interference (RNAi) (Ralph et al. (2005) Nat. Medicine 11: 429-433). The mechanism of RNAi involves the processing of long dsRNA into double-stranded, approximately 21-25 nucleotide (nt) RNA. These products are called small interfering or silencing RNAs (siRNAs), which are sequence-specific mediators of mRNA degradation. In differentiated mammalian cells, dsRNA >30 bp has been shown to activate an interferon response that results in the shutdown of protein synthesis and nonspecific mRNA degradation (Stark et al. (1998) Ann. Rev. Biochem. 67: 227-64). However, it is possible to circumvent this response by using 21 nt siRNA duplexes (Elbashir et al. (2001) EMBO J. 20: 6877-88; Hutvagner et al. (2001) Science 293: 834-8), allowing gene function to be analyzed in cultured mammalian cells.

[0468] shRNAs consist of short inverted RNA repeats separated by small loop sequences that are rapidly processed by the cellular machinery into 19-22 nt siRNAs that silence target gene expression.

[0469] MicroRNAs (miRNAs) are small (22-25 nucleotides long) non-coding RNAs that can effectively reduce the translation of target mRNAs by binding to their 3' untranslated regions (UTRs). MicroRNAs are a very large group of small RNAs that are naturally produced in organisms and at least in part regulate the expression of target genes. The first members of the microRNA family are let-7 and lin-4. The let-7 gene encodes a highly conserved small RNA species that regulates the expression of endogenous protein-coding genes during nematode development. The active RNA species is initially transcribed as a ∼70 nt precursor that is post-transcriptionally processed to a mature ∼21 nt form. Both let-7 and lin-4 are transcribed as hairpin RNA precursors that are processed to their mature forms by the Dicer enzyme.

[0470] The concept of antisense is the selective binding of small, possibly modified, DNA or RNA molecules to messenger RNA in cells, preventing synthesis of the encoded protein.

[0471] Methods for designing siRNA, shRNA, miRNA and antisense DNA / RNA to regulate the expression of target proteins, as well as methods for delivering these substances to cells of interest, are well known in the art.

[0472] Adenovirus Proteins Adenoviruses are natural co-helpers for AAV infection and provide a set of genes that optimize AAV infection: E1a, E1b, E2a and E4.

[0473] It has been previously demonstrated that delivery of adenoviral proteins during gene editing improves the efficiency of HDR in HSCs (and promotes long-term repopulating activity of HSCs). Without wishing to be bound by theory, adenoviral proteins may provide a helper function in AAV infection during gene editing.

[0474] In some embodiments, one or more senescence inhibitors (eg, a MAPK inhibitor, an IL-1 inhibitor, and / or an NF-κB inhibitor) are used in combination with at least one adenoviral protein.

[0475] In one embodiment, the one or more senescence inhibitors (eg, a MAPK inhibitor, an IL-1 inhibitor, and / or an NF-κB inhibitor) comprise at least one adenoviral protein.

[0476] In one embodiment, the agent that promotes homologous sequence-dependent DNA repair comprises at least one adenoviral protein.

[0477] In one embodiment, the one or more senescence inhibitors (eg, a MAPK inhibitor, an IL-1 inhibitor, and / or an NF-κB inhibitor) comprise a nucleic acid sequence encoding at least one adenoviral protein.

[0478] In one embodiment, the agent that promotes homologous sequence-dependent DNA repair comprises a nucleic acid sequence encoding at least one adenoviral protein.

[0479] The adenoviral protein is not limited to a particular adenovirus serotype. For example, in one embodiment, the at least one adenoviral protein is derived from an adenovirus of serotype 4, an adenovirus of serotype 5, an adenovirus of serotype 7, and / or an adenovirus of serotype 9.

[0480] In one embodiment, the at least one adenoviral protein is selected from the group including E1a, E1b, E2a and E4.

[0481] In a preferred embodiment, the at least one adenoviral protein is the open reading frame of the E4 gene.

[0482] In a preferred embodiment, the at least one adenoviral protein is E4orf1 or a variant thereof.

[0483] An example of a nucleotide sequence encoding Ad5-E4orf1 is shown in SEQ ID NO: 10. Suitably, at least one adenoviral protein may comprise the nucleotide sequence for mRNA translation shown in SEQ ID NO: 10 or a variant thereof. TIFF2024544746000022.tif22165TIFF2024544746000023.tif12165

[0484] An example of the amino acid sequence of Ad5-E4orf1 is shown in SEQ ID NO: 1. Suitably, at least one adenoviral protein may comprise the amino acid sequence shown in SEQ ID NO: 1, or a variant thereof. TIFF2024544746000024.tif16165

[0485] Other examples of the amino acid sequence of E4orf1 are shown in SEQ ID NO: 57 to SEQ ID NO: 76. Preferably, at least one adenoviral protein may comprise an amino acid sequence shown in SEQ ID NO: 57 to SEQ ID NO: 76, or a variant thereof.

[0486] TIFF2024544746000025.tif164168TIFF2024544746000026.tif243168

[0487] In a preferred embodiment, the at least one adenoviral protein is E4orf6 / 7 or a variant thereof.

[0488] An example of a nucleotide sequence encoding Ad5-E4orf6 / 7 is shown in SEQ ID NO: 11. Suitably, at least one adenoviral protein may comprise a nucleotide sequence for mRNA translation shown in SEQ ID NO: 11, or a variant thereof. TIFF2024544746000027.tif37163

[0489] An example of the amino acid sequence of Ad5-E4orf6 / 7 is shown in SEQ ID NO: 2. Suitably, at least one adenoviral protein may comprise the amino acid sequence shown in SEQ ID NO: 2, or a variant thereof. TIFF2024544746000028.tif17163

[0490] Other examples of amino acid sequences of E4orf6 / 7 are shown in SEQ ID NO: 7, SEQ ID NO: 39 to 56, and SEQ ID NO: 77 to 88. Suitably, at least one adenoviral protein may comprise the amino acid sequence shown in SEQ ID NO: 7, SEQ ID NO: 39 to 56, and SEQ ID NO: 77 to 88 or a variant thereof.

[0491] TIFF2024544746000029.tif77166TIFF2024544746000030.tif243155TIFF20245447460 00031.tif243154TIFF2024544746000032.tif243155TIFF2024544746000033.tif37156

[0492] An example of a nucleotide sequence encoding E4orf6 is shown in SEQ ID NO:12. TIFF2024544746000034.tif72165

[0493] An example of the amino acid sequence of E4orf6 is shown in SEQ ID NO:13. TIFF2024544746000035.tif27166

[0494] An example of a nucleotide sequence encoding E1B55K is shown in SEQ ID NO:14. TIFF2024544746000036.tif62166TIFF2024544746000037.tif58164

[0495] An example of the amino acid sequence of E1B55K is shown in SEQ ID NO:15. TIFF2024544746000038.tif43164

[0496] In one embodiment, the at least one adenoviral protein is not E4orf6. In one embodiment, the at least one adenoviral protein is not E1B55K. In one embodiment, the at least one adenoviral protein does not include E4orf6 or E1B55K.

[0497] Preferably, the at least one adenoviral protein is E4ORF1, and preferably the amino acid sequence of E4ORF1 is set forth in SEQ ID NO: 1, or SEQ ID NOs: 57 to 76. The at least one adenoviral protein may be a variant or fragment of E4ORF1, provided that it substantially retains the biological activity of full-length E4ORF1, e.g., the ability to increase survival and / or engraftment of gene-edited hematopoietic stem and / or progenitor cells as defined herein.

[0498] Preferably, the at least one adenoviral protein is E4ORF6 / 7, and preferably the amino acid sequence of E4ORF6 / 7 is set forth in SEQ ID NO: 2, or SEQ ID NOs: 77 to 107. The at least one adenoviral protein may be a variant or fragment of E4ORF6 / 7, provided that it substantially retains the biological activity of full-length E4ORF6 / 7, e.g., the ability to increase survival and / or engraftment of gene-edited hematopoietic stem and / or progenitor cells as defined herein.

[0499] In one embodiment, the drug comprises adenoviral protein E4ORF1, preferably wherein the amino acid sequence of E4ORF1 is shown in SEQ ID NO: 1, or SEQ ID NO: 57 to 76; and adenoviral protein E4ORF6 / 7, preferably wherein the amino acid sequence of E4ORF6 / 7 is shown in SEQ ID NO: 2, or SEQ ID NO: 77 to 107.

[0500] In one embodiment, the inhibitor of the present invention or the agent for use of the present invention comprises a nucleic acid sequence encoding the adenoviral protein E4ORF1, preferably whose amino acid sequence of E4ORF1 is shown in SEQ ID NO: 1 or SEQ ID NO: 57 to 76; and a nucleic acid sequence encoding the adenoviral protein E4ORF6 / 7, preferably whose amino acid sequence of E4ORF6 / 7 is shown in SEQ ID NO: 2 or SEQ ID NO: 77 to 107.

[0501] In one embodiment, the agent comprises two compounds as defined herein that promote homologous sequence-dependent repair.In one embodiment, the agent comprises an inhibitor of p53 activation and an adenovirus protein or its coding nucleotide sequence.Preferably, the inhibitor of p53 activation is GSE56 or its variant.

[0502] In one embodiment, the medicament comprises: (a) an inhibitor of p53 activation and an adenoviral protein E4ORF1 (or a nucleotide sequence encoding same). In one embodiment, E4ORF1 has the amino acid sequence of E4ORF1 set forth in SEQ ID NO: 1, or SEQ ID NOs: 57-76; (b) an inhibitor of p53 activation and an adenoviral protein E4ORF6 / 7 (or a nucleotide sequence encoding same). In one embodiment, E4ORF6 / 7 has the amino acid sequence of E4ORF6 / 7 shown in SEQ ID NO: 2, or SEQ ID NOs: 77 to 107. (c) an inhibitor of p53 activation, preferably an adenoviral protein E4ORF1 (or a nucleotide sequence encoding same) whose amino acid sequence of E4ORF1 is shown in SEQ ID NO: 1 or SEQ ID NO: 57 to 76; and an adenoviral protein E4ORF6 / 7 (or a nucleotide sequence encoding same) whose amino acid sequence of E4ORF6 / 7 is shown in SEQ ID NO: 2 or SEQ ID NO: 77 to 107. Preferably, the inhibitor of p53 activation is GSE56 or a variant thereof.

[0503] In one embodiment, the agent is a composition comprising an inhibitor of p53 activation and an adenoviral protein, or a nucleotide sequence encoding same.

[0504] In one embodiment, the inhibitor of p53 activation is administered in combination with the adenoviral protein or nucleotide sequence encoding same, either simultaneously, sequentially or separately.

[0505] In one embodiment, the adenoviral protein or the nucleotide sequence encoding it is administered in combination with an inhibitor of p53 activation either simultaneously, sequentially or separately.

[0506] In one embodiment, the nucleic acid encoding an adenoviral protein is an mRNA.

[0507] In one embodiment, the adenoviral proteins are transiently expressed in hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells. Preferably, the transient expression occurs during gene editing and / or transduction of the hematopoietic cells, hematopoietic stem cells, progenitor cells and / or T cells.

[0508] combination The present invention encompasses all possible combinations of the embodiments described herein, regardless of whether the embodiments are specifically described as such.

[0509] In one embodiment, the present invention provides one or more aging inhibitors in combination with one or more of the following: (i) one or more adenoviral proteins described herein, or a nucleic acid encoding one or more adenoviral proteins; (ii) one or more transduction enhancers as described herein; (iii) one or more p53 inhibitors as described herein; and / or (iv) Integrase-deficient lentiviral vector (IDLV).

[0510] In another embodiment, the present invention provides one or more aging inhibitors in combination with: (i) one or more adenoviral proteins described herein, or a nucleic acid encoding one or more adenoviral proteins; (ii) one or more transduction enhancers as described herein; (iii) one or more p53 inhibitors described herein; and (iv) Integrase-deficient lentiviral vector (IDLV).

[0511] In a preferred embodiment, the P53 inhibitor is CsH.

[0512] In another preferred embodiment, the transduction enhancer is CsH.

[0513] In a preferred embodiment, the present invention provides one or more aging inhibitors in combination with: (i) one or more adenoviral proteins described herein, or a nucleic acid encoding one or more adenoviral proteins; (ii) CsH; (iii) GSE56; and (iv) Integrase-deficient lentiviral vector (IDLV).

[0514] These combinations are specifically disclosed with respect to all of the senescence inhibitors, uses, methods, pharmaceutical compositions, and gene-edited and / or transduced populations of hematopoietic cells of the present invention.

[0515] Isolation and enrichment of cell populations The term "isolated population" of cells, as used herein, may refer to a population of cells previously removed from the body. The isolated population of cells may be cultured and manipulated ex vivo or in vitro using standard techniques known in the art. The isolated population of cells may later be reintroduced into a subject, which may be the same subject from which the cells were originally isolated or a different subject.

[0516] A cell population can be purified selectively for cells that exhibit a particular phenotype or characteristic from other cells that do not, or exhibit to a lesser extent, that phenotype or characteristic. For example, a cell population that expresses a particular marker (such as CD34) can be purified from a starting cell population. Alternatively, or in addition, a cell population that does not express another marker (such as CD38) can be purified.

[0517] "Enriching" a cell population for a particular cell type should be understood as increasing the concentration of that cell type within the population. The concentrations of other cell types may be concomitantly decreased.

[0518] Purification or enrichment can result in a nearly pure cell population, free of other cell types.

[0519] Purification or enrichment for a cell population expressing a specific marker (e.g., CD34 or CD38) can be achieved by using an agent that binds to that marker, preferably an agent that binds substantially specifically to that marker.

[0520] The agent that binds to a cell marker may be an antibody, such as an anti-CD34 or anti-CD38 antibody.

[0521] The term "antibody" refers to intact antibodies or antibody fragments capable of binding to a selected target, and includes Fv, ScFv, F(ab') and F(ab')2, monoclonal and polyclonal antibodies, engineered antibodies, including chimeric antibodies, CDR-grafted antibodies and humanized antibodies, as well as artificially selected antibodies produced using phage display or other techniques.

[0522] In addition, alternatives to classical antibodies, such as "avibodies", "avimers", "anticalins", "nanobodies" and "DARPins", can also be used in the present invention.

[0523] The agent that binds to a specific marker can be labeled so as to be identifiable using any of a number of techniques known in the art. The agent may be inherently labeled or may be modified by conjugating a label to it. By "conjugate", it should be understood that the agent and the label are functionally linked. This means that the agent and the label are linked to each other so that both can perform their functions (e.g., bind to the marker, allow fluorescent identification, or allow separation when placed in a magnetic field) substantially without hindrance. Suitable methods of conjugation are well known in the art and can be easily identified by those skilled in the art.

[0524] The label can, for example, allow the labeled agent and any cells to which it is bound to be purified from their environment (e.g., the agent can be labeled with magnetic beads, or affinity tags such as avidin), detected, or both. Detectable markers suitable for use as labels include fluorophores (e.g., green, cherry, cyan, and orange fluorescent proteins) and peptide tags (e.g., His tags, Myc tags, FLAG tags, and HA tags).

[0525] Numerous techniques are known in the art for isolating cell populations expressing specific markers. These include magnetic bead-based separation techniques (e.g., closed-loop magnetic bead-based separation), flow cytometry, fluorescence activated cell sorting (FACS), affinity tag purification (e.g., affinity columns or beads, e.g., biotin columns for separating avidin-labeled drugs), and microscopy-based techniques.

[0526] Separation can also be performed using a combination of different techniques, for example a magnetic bead-based separation step followed by selection of the resulting cell population for one or more additional (positive or negative) markers by flow cytometry.

[0527] Clinical grade separations can be performed, for example, using the CliniMACS® system (Miltenyi), which is an example of a closed-circuit magnetic bead-based separation technology.

[0528] It is also envisioned that dye exclusion properties (eg, side population or rhodamine labeling) or enzymatic activity (eg, ALDH activity) can be used to enrich for hematopoietic stem cells.

[0529] Preferably, the agent reduces the expression of CD34 in the population of gene-edited cells compared to a population of untreated gene-edited cells. + CD133 + CD90 + Does not reduce the cellular fraction.

[0530] Gene editing The term "gene editing" refers to a type of genetic manipulation in which nucleic acid is inserted, deleted, or replaced in a cell. Gene editing can be achieved using engineered nucleases, which can be targeted to desired sites in polynucleotides (e.g., genomes). Such nucleases can generate site-specific double-strand breaks at desired locations, which can then be repaired through non-homologous end joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations.

[0531] Such nucleases can be delivered to target cells using viral vectors.The present invention provides methods to increase the efficiency of the gene editing process.

[0532] Examples of suitable nucleases known in the art include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) / Cas systems (Gaj, T. et al. (2013) Trends Biotechnol. 31: 397-405; Sander, JD et al. (2014) Nat. Biotechnol. 32: 347-55). Meganucleases (Silve, G. et al. (2011) Cur. Gene Ther. 11: 11-27) can also be used as suitable nucleases for gene editing.

[0533] The CRISPR / Cas system is an RNA-guided DNA-binding system (van der Oost et al. (2014) Nat. Rev. Microbiol. 12: 479-92) in which a guide RNA (gRNA) can be selected to enable the Cas9 domain to target a specific sequence. Methods for designing gRNAs are known in the art. Furthermore, fully orthogonal Cas9 proteins have been developed in recent years, as well as Cas9 / gRNA ribonucleoprotein complexes and modifications of gRNA structure / composition to bind different proteins in order to simultaneously and directionally target different effector domains to desired genomic sites in cells (Esvelt et al. (2013) Nat. Methods 10: 1116-21; Zetsche, B. et al. (2015) Cell pii: S0092-8674(15)01200-3; Dahlman, JE et al. (2015) Nat. Biotechnol. 2015 Oct 5. doi: 10.1038 / nbt.3390. [Epub ahead of print]; Zalatan, JG et al. (2015) Cell 160: 339-50; Paix, A. et al. (2015) Genetics 201: 47-54), are suitable for use in the present invention.

[0534] Pretreatment The method of the present invention may further include a pre-culture step. As used herein, the term "pre-culture step" may refer to a culture step carried out before the introduction of a gene editing mechanism into a cell population and / or the transduction of a cell population. As used herein, the term "pre-culture step" may refer to an activation step or a stimulation step carried out before the introduction of a gene editing mechanism into a cell population and / or the transduction of a cell population. As used herein, the term "pre-expansion step" may refer to an expansion step carried out before the introduction of a gene editing mechanism into a cell population and / or the transduction of a cell population.

[0535] In some embodiments, the method further comprises a pre-culturing step prior to contacting the cell population with one or more senescence inhibitors. In some embodiments, the method further comprises a pre-culturing step prior to and / or during contacting the cell population with the one or more senescence inhibitors. In some embodiments, the method further comprises a pre-culturing step prior to introducing a gene editing mechanism into the cell population. In some embodiments, the method further comprises a pre-culturing step prior to transducing the cell population.

[0536] The pre-culture step (eg, pre-activation step and / or pre-expansion step) may be carried out using any suitable conditions.

[0537] During the pre-culture step (e.g., pre-activation step and / or pre-expansion step), the cell population is approximately 1×10 5 cells / mL ~ approx. 10×10 5 Cells / mL, e.g., approximately 2 x 10 5 cells / mL, or approximately 5 x 10 5 Cells may be seeded at a concentration of 1000 cells / mL.

[0538] Preferably, the pre-culture step (e.g., pre-activation and / or pre-expansion step) is for at least 1 day, at least 2 days, or at least 3 days. Preferably, the cell population is pre-cultured (e.g., pre-activation and / or pre-expansion) for about 3 days. Preferably, the cell population is pre-cultured at 37° C. in a humidified atmosphere of 5% CO2.

[0539] Any suitable culture medium may be used. For example, commercially available media such as StemSpan medium may be used, which contains bovine serum albumin, insulin, transferrin, and Iscove's MDM supplements. The culture medium may be supplemented with one or more antibiotics (e.g., penicillin, streptomycin).

[0540] The pre-culture step (e.g., pre-activation step and / or pre-expansion step) can be performed in the presence of one or more cytokines and / or growth factors. As used herein, a "cytokine" is any cell signaling substance, including chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. As used herein, a "growth factor" is any substance capable of stimulating cell proliferation, wound healing, or cell differentiation. The terms "cytokine" and "growth factor" can overlap.

[0541] The pre-culture step (eg, pre-activation step and / or pre-expansion step) can be carried out in the presence of one or more early acting cytokines, one or more transduction enhancing agents, and / or one or more expansion enhancing agents.

[0542] Early acting cytokines As used herein, an "early acting cytokine" is a cytokine that stimulates cells such as HSCs or HPCs. Early acting cytokines include thrombopoietin (TPO), stem cell factor (SCF), Flt3-ligand (FLT3-L), interleukin (IL)-3, and IL-6. In some embodiments, the pre-culture step (e.g., pre-activation step and / or pre-expansion step) is performed in the presence of at least one early acting cytokine. Any suitable concentration of the early acting cytokine may be used. For example, 1-1000 ng / mL, or 10-1000 ng / mL, or 10-500 ng / mL.

[0543] In some embodiments, the pre-culture step (e.g., the pre-activation step and / or the pre-expansion step) is performed in the presence of SCF. The concentration of SCF may be about 10 to 1000 ng / mL, about 50 to 500 ng / mL, or about 100 to 300 ng / mL.

[0544] In some embodiments, the pre-culture step (e.g., the pre-activation step and / or the pre-expansion step) is carried out in the presence of FLT3-L. The concentration of FLT3-L may be about 10 to 1000 ng / mL, about 50 to 500 ng / mL, or about 100 to 300 ng / mL.

[0545] In some embodiments, the pre-culture step (e.g., the pre-activation step and / or the pre-expansion step) is carried out in the presence of TPO. The concentration of TPO may be about 5 to 500 ng / mL, about 10 to 200 ng / mL, or about 20 to 100 ng / mL.

[0546] In some embodiments, the pre-culture step (e.g., the pre-activation step and / or the pre-expansion step) is performed in the presence of IL-3. The concentration of IL-3 can be about 10 to 200 ng / mL, about 20 to 100 ng / mL, or about 60 ng / mL.

[0547] In some embodiments, the pre-culture step (e.g., the pre-activation step and / or the pre-expansion step) is performed in the presence of IL-6. The concentration of IL-6 can be about 5 to 100 ng / mL, about 10 to 50 ng / mL, or about 20 ng / mL.

[0548] In some embodiments, the pre-culture step (e.g., pre-activation step and / or pre-expansion step) is particularly useful for determining whether the cell population is cord blood CD34 + In the case of cells, the treatment is performed in the presence of SCF (e.g., at a concentration of about 100 ng / mL), FLT3-L (e.g., at a concentration of about 100 ng / mL), TPO (e.g., at a concentration of about 20 ng / mL) and IL-6 (e.g., at a concentration of about 20 ng / mL).

[0549] In some embodiments, the pre-culture step (e.g., pre-activation step and / or pre-expansion step) is particularly useful in cases where the cell population is (mobilized) peripheral blood CD34 + In the case of cells, the treatment is performed in the presence of SCF (e.g., at a concentration of about 300 ng / mL), FLT3-L (e.g., at a concentration of about 300 ng / mL), TPO (e.g., at a concentration of about 100 ng / mL) and IL-3 (e.g., at a concentration of about 60 ng / mL).

[0550] Transduction Enhancers As used herein, a "transduction enhancing agent" is a substance that can improve viral transduction of cells, such as HSCs or HPCs. Suitable transduction enhancing agents include LentiBOOST, prostaglandin E2 (PGE2), protamine sulfate (PS), Vectofusin-1, ViraDuctin, RetroNectin, Staurosporine (Stauro), 7-hydroxy-stauro, human serum albumin, polyvinyl alcohol, and cyclosporine H (CsH). In some embodiments, the pre-culture step (e.g., pre-activation step and / or pre-expansion step) is performed in the presence of at least one transduction enhancing agent. Any suitable concentration of the transduction enhancing agent can be used, for example, as described in Schott, JW, et al., 2019. Molecular Therapy-Methods & Clinical Development, 14, pp.134-147 or Yang, H., et al., 2020. Molecular Therapy-Nucleic Acids, 20, pp. 451-458.

[0551] In some embodiments, the pre-culture step (e.g., the pre-activation step and / or the pre-expansion step) is carried out in the presence of PGE2. Preferably, the PGE2 is 16,16-dimethyl prostaglandin E2 (dmPGE2). The concentration of PGE2 may be about 1 to 100 μM, about 5 to 20 μM, or about 10 μM.

[0552] In some embodiments, the pre-culture step (e.g., the pre-activation step and / or the pre-expansion step) is carried out in the presence of CsH. The concentration of CsH can be about 1-50 μM, 5-50 μM, about 10-50 μM, or about 10 μM.

[0553] Expansion promoter As used herein, an "expansion promoter" is a substance that can improve the expansion of cells such as HSCs or HPCs. Suitable expansion promoters include UM171, UM729, StemRegenin1 (SR1), diethylaminobenzaldehyde (DEAB), LG1506, BIO (GSK3β inhibitor), NR-101, trichostatin A (TSA), garcinol (GAR), valproic acid (VPA), copper chelator, tetraethylenepentamine, and nicotinamide. In some embodiments, the pre-culture step (e.g., pre-activation step and / or pre-expansion step) is performed in the presence of at least one expansion promoter. As described in Huang, X., et al., 2019. F1000Research, 8, 1833, any suitable concentration of the expansion promoter can be used.

[0554] In some embodiments, the pre-culture step (e.g., the pre-activation step and / or the pre-expansion step) is carried out in the presence of UM171 or UM729. The concentration of UM171 can be about 10-200 nM, about 20-100 nM, or about 50 nM.

[0555] In some embodiments, the pre-culture step (e.g., the pre-activation step and / or the pre-expansion step) is carried out in the presence of SR1. The concentration of SR1 can be about 0.1 to 10 μM, about 0.5 to 5 μM, or about 1 μM.

[0556] In some embodiments, the pre-culture step (e.g., pre-activation step and / or pre-expansion step) is performed in the presence of UM171 (e.g., at a concentration of about 50 nM) or UM729 and SR1 (e.g., at a concentration of about 1 μM).

[0557] In some embodiments, the pre-culture step (e.g., pre-activation step and / or pre-expansion step) is particularly useful for determining whether the cell population is cord blood CD34 +In the case of cells, the treatment is performed in the presence of SCF (e.g., at a concentration of about 100 ng / mL), FLT3-L (e.g., at a concentration of about 100 ng / mL), TPO (e.g., at a concentration of about 20 ng / mL), IL-6 (e.g., at a concentration of about 20 ng / mL), PGE2 (e.g., at a concentration of about 10 μM), UM171 (e.g., at a concentration of about 50 nM), and SR1 (e.g., at a concentration of about 1 μM).

[0558] In some embodiments, the pre-culture step (e.g., pre-activation step and / or pre-expansion step) is particularly useful in cases where the cell population is (mobilized) peripheral blood CD34 + In the case of cells, the treatment is performed in the presence of SCF (e.g., at a concentration of about 300 ng / mL), FLT3-L (e.g., at a concentration of about 300 ng / mL), TPO (e.g., at a concentration of about 100 ng / mL), IL-3 (e.g., at a concentration of about 60 ng / mL), PGE2 (e.g., at a concentration of about 10 μM), UM171 (e.g., at a concentration of about 50 nM), and SR1 (e.g., at a concentration of about 1 μM).

[0559] vector A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. The vectors used to transduce hematopoietic stem and / or progenitor cells in the present invention are viral vectors.

[0560] Preferably, the viral vector is in the form of a viral vector particle.

[0561] The viral vector may be, for example, an adeno-associated viral (AAV), adenoviral, retroviral or lentiviral vector.Preferably, the viral vector is an AAV vector or a retroviral or lentiviral vector, more preferably an AAV vector.Preferably, the retroviral vector is not a gamma-retroviral vector.

[0562] A "vector derived from" a particular type of virus should be understood to mean that the vector contains at least one component portion that is derivable from that type of virus.

[0563] Adeno-associated virus (AAV) vectors Adeno-associated virus (AAV) is an attractive vector system for use in the present invention because it has a high frequency of integration and can infect non-dividing cells, making it useful for delivery of genes to mammalian cells in tissue culture.

[0564] AAV has a broad host range of infectious properties. Details regarding the construction and use of AAV vectors are described in U.S. Patent No. 5,139,941 and U.S. Patent No. 4,797,368.

[0565] Recombinant AAV vectors have been used successfully for the in vitro and in vivo transduction of marker genes and genes involved in human diseases.

[0566] Preferred vectors are those that can achieve high transduction efficiency in primary human cells, such as HSPC cells.

[0567] In one embodiment, the vector is an AAV6 vector or a vector derived from an AAV6 vector. Preferably, the vector is an AAV6 vector.

[0568] Adenovirus Vectors Adenoviruses are double-stranded, linear DNA viruses that do not undergo an RNA intermediate. There are over 50 different human serotypes of adenovirus, classified into six subgroups based on genetic sequence homology. The natural target of adenoviruses is the respiratory and gastrointestinal epithelium, and generally results in only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are the most commonly used in adenovirus vector systems, and are usually associated with upper respiratory tract infections in young people.

[0569] Adenoviruses have been used as vectors for gene therapy and for the expression of heterologous genes. The large (36 kb) genome can accommodate up to 8 kb of foreign DNA insert and replicates efficiently in complementation cell lines, resulting in up to 10 12 Very high titers of adenovirus can be generated, therefore, adenoviruses are one of the best systems to study gene expression in primary replication-incompetent cells.

[0570] Expression of viral or foreign genes from the adenoviral genome does not require a replicating cell. Adenoviral vectors enter cells by receptor-mediated endocytosis. Once inside the cell, adenoviral vectors rarely integrate into host chromosomes. Instead, they function episomally (independently of the host genome) like a linear genome in the host nucleus. Thus, the use of recombinant adenoviruses alleviates the problems associated with random integration into the host genome.

[0571] Retroviral and lentiviral vectors Retroviral vectors may be or may be derived from any suitable retrovirus. Many different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myeloblastosis virus-29 (MC29) and avian erythroblastosis virus (AEV). A detailed list of retroviruses can be found in Coffin, JM et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63.

[0572] Retroviruses can be broadly divided into two categories: "simple" and "complex". Retroviruses can be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are lentiviruses and spumaviruses. A review of these retroviruses is given in Coffin, JM et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63.

[0573] The basic structure of the genome of retroviruses and lentiviruses shares many common features, such as 5' LTR and 3' LTR. Located between or within these are a packaging signal that allows packaging of the genome, a primer binding site, an integration site that allows integration into the host cell genome, and the gag, pol, and env genes that code for packaging components, which are polypeptides necessary for the construction of viral particles. Lentiviruses have additional features, such as the rev and RRE sequences in HIV, which allow efficient transport of the RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of the infected target cell.

[0574] In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for the integration and transcription of the provirus. The LTRs can also act as enhancer-promoter sequences and control the expression of viral genes.

[0575] The LTRs are themselves identical sequences that can be divided into three elements, U3, R and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of these three elements can vary considerably between different retroviruses.

[0576] In defective retroviral vector genomes, gag, pol and env may be absent or non-functional.

[0577] In a typical retroviral vector, at least a portion of one or more protein coding regions essential for replication can be removed from the virus, rendering the viral vector replication-deficient. Also, portions of the viral genome can be replaced with libraries encoding candidate regulatory moieties operably linked to regulatory control regions and reporter moieties within the vector genome to generate vectors comprising candidate regulatory moieties capable of transducing target host cells and / or integrating their genome into the host genome.

[0578] Lentiviral vectors are part of a larger group of retroviral vectors. A detailed list of lentiviruses can be found in Coffin, JM et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. In summary, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the etiological agent of human acquired immune deficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype "slow virus" Visna / Maedi virus (VMV), as well as the related Caprine Arthritis Encephalomyelitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), and the more recently described Feline Immunodeficiency Virus (FIV) and Bovine Immunodeficiency Virus (BIV).

[0579] The lentivirus family differs from retroviruses in that lentiviruses are capable of infecting both dividing and non-dividing cells (Lewis, P et al. (1992) EMBO J. 11: 3053-8; Lewis, PF et al. (1994) J. Virol. 68: 510-6). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells, such as those that make up, for example, muscle, brain, lung and liver tissue.

[0580] A lentiviral vector, as used herein, is a vector that comprises at least one component that can be derived from a lentivirus, preferably the component that is involved in the biological mechanisms by which the vector infects cells and expresses genes or is replicated.

[0581] The lentiviral vector may be a "primate" vector. The lentiviral vector may be a "non-primate" vector (i.e., derived from a virus that does not primarily infect primates, especially humans). An example of a non-primate lentivirus may be any member of the lentiviridae family that does not naturally infect primates.

[0582] As examples of lentivirus-based vectors, HIV-1 and HIV-2 based vectors are described below.

[0583] HIV-1 vectors contain cis-acting elements that are also found in simple retroviruses. Sequences extending into the gag open reading frame have been shown to be important for HIV-1 packaging. Thus, HIV-1 vectors often contain relevant portions of gag with mutated translation initiation codons. In addition, most HIV-1 vectors also contain a portion of the env gene, including the RRE. Rev binds to the RRE, allowing transport of full-length or singly spliced ​​mRNAs from the nucleus to the cytoplasm. In the absence of Rev and / or RRE, full-length HIV-1 RNA accumulates in the nucleus. Alternatively, constitutive transport elements from certain simple retroviruses, such as the Mason-Pfizer monkey virus, can be used to eliminate the need for Rev and RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

[0584] Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Like HIV-1-based vectors, HIV-2 vectors also require the RRE for efficient transport of full-length or singly spliced ​​viral RNA.

[0585] In one system, when vector and helper construct are derived from two different viruses, the reduced nucleotide homology may reduce the probability of recombination.In addition to primate lentivirus-based vectors, FIV-based vectors have also been developed as an alternative to vectors derived from pathogenic HIV-1 genome.The structure of these vectors is also similar to that of HIV-1-based vectors.

[0586] Preferably, the viral vectors used in the present invention have a minimal viral genome.

[0587] "Minimal viral genome" should be understood to mean that the viral vector has been engineered to remove non-essential elements and retain essential elements to provide the functions required to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO1998 / 017815.

[0588] Preferably, the plasmid vector used to produce the viral genome in the host / packaging cell has sufficient lentiviral genetic information to allow packaging of the RNA genome into viral particles capable of infecting target cells in the presence of packaging components, but is incapable of independent replication to produce infectious viral particles in the final target cell. Preferably, the vector lacks functional gag-pol and / or env genes and / or other genes essential for replication.

[0589] However, the plasmid vectors used to produce the viral genome in the host cell / packaging cell also contain transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the host cell / packaging cell. These regulatory sequences may be the native sequences associated with the transcribed viral sequences (i.e., the 5' U3 region) or they may be a heterologous promoter, such as another viral promoter (e.g., the CMV promoter).

[0590] The vector may be a self-inactivating (SIN) vector in which viral enhancer and promoter sequences have been deleted. SIN vectors can be generated in vivo and transduce non-dividing cells with efficacy comparable to that of wild-type vectors. Transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should avoid recruitment by replication-competent viruses. This should also allow regulated expression of genes from internal promoters by eliminating the cis-acting effects of the LTR.

[0591] The vector may be integration-defective. Integration-defective lentiviral vectors (IDLV) can be produced, for example, by packaging the vector with a catalytically inactive integrase (such as HIV integrase with D64V mutation at the catalytic site; Naldini, L. et al. (1996) Science 272: 263-7; Naldini, L. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 11382-8; Leavitt, AD et al. (1996) J. Virol. 70: 721-8), or by modifying or deleting essential att sequences from the vector LTR (Nightingale, SJ et al. (2006) Mol. Ther. 13: 1121-32), or by a combination of the above.

[0592] Target locus In one embodiment, gene editing targets a hematopoietic stem and / or progenitor cell locus. In one embodiment, the donor template targets a hematopoietic stem and / or progenitor cell locus.

[0593] In one embodiment, the gene editing targets a T cell locus. In one embodiment, the donor template targets a T cell locus.

[0594] AAVS locus In one embodiment, the adeno-associated virus integration site 1 (AAVS1) locus is targeted. In one embodiment, the donor template targets the adeno-associated virus integration site 1 (AAVS1) locus.

[0595] An example of an AAV donor cassette for AAVS1 can include the following nucleotide sequence: TIFF2024544746000039.tif156164TIFF2024544746000040.tif168166

[0596] IL2RG locus In another embodiment, gene editing targets interleukin 2 receptor subunit gamma (IL2RG), preferably intron 1 of IL2RG. In another embodiment, the donor template targets interleukin 2 receptor subunit gamma (IL2RG), preferably intron 1 of IL2RG.

[0597] An example of an AAV donor cassette for IL2RG can include the following nucleotide sequence: TIFF2024544746000041.tif224165TIFF2024544746000042.tif242164TIFF2024544746000043.tif89165

[0598] In another embodiment, the gene editing targets RAG-1. In another embodiment, the donor template targets RAG-1.

[0599] Nucleotide of interest A vector for use in the present invention preferably comprises one or more nucleotides of interest.

[0600] Preferably, the subject nucleotides provide a therapeutic effect.

[0601] Suitably, the one or more NOIs for use in the present invention may be selected from a guide RNA, a nucleotide sequence encoding a Cas9 ribonucleoprotein, a nucleotide sequence encoding one or more adenoviral proteins, a nucleotide sequence encoding an agent that promotes homologous sequence dependent DNA repair (e.g. an inhibitor of p53 activation or a nucleotide sequence encoding one or more adenoviral proteins).

[0602] Suitable NOIs include, but are not limited to, sequences encoding enzymes, cytokines, chemokines, hormones, antibodies, antioxidant molecules, engineered immunoglobulin-like molecules, single chain antibodies, fusion proteins, immune co-stimulatory molecules, immune modulatory molecules, antisense RNA, microRNA, shRNA, siRNA, guide RNA (gRNA, e.g. used in conjunction with the CRISPR / Cas system), ribozymes, miRNA target sequences, trans domain negative mutants of target proteins, toxins, conditional toxins, antigens, tumor suppressor proteins, growth factors, transcription factors, membrane proteins, surface receptors, anti-cancer molecules, vasoactive proteins and peptides, antiviral proteins and ribozymes, and derivatives thereof (e.g. derivatives with associated reporter groups). The NOI may also encode a prodrug activating enzyme. Preferably, the NOI is a guide RNA (gRNA).

[0603] Pharmaceutical Compositions In one embodiment, the cells of the invention can be formulated with a pharma- ceutically acceptable carrier, diluent or excipient for administration to a subject. Suitable carriers and diluents include isotonic saline, such as phosphate buffered saline, and may contain human serum albumin.

[0604] Handling of cell therapy products is preferably carried out in accordance with the FACT-JACIE International Code for Cell Therapy.

[0605] Transplantation of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells The invention provides populations of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells prepared according to the methods of the invention for use in therapy, for example for use in gene therapy.

[0606] The use may be part of a cell transplantation procedure, such as a hematopoietic stem cell transplantation procedure.

[0607] Hematopoietic stem cell transplantation (HSCT) is the transplantation of blood stem cells derived from bone marrow (in this case known as bone marrow transplant) or blood. Stem cell transplantation is the most commonly performed medical procedure in the fields of hematology and oncology for people with blood or bone marrow diseases or certain types of cancer.

[0608] Many recipients of HSCT are multiple myeloma or leukemia patients who may not benefit from long-term treatment with chemotherapy or who are already resistant to chemotherapy. Candidates for HSCT include pediatric cases in which patients have congenital defects such as severe combined immunodeficiency or congenital neutropenia with defective stem cells, and also children or adults with aplastic anemia who have lost stem cells after birth. Other conditions treated with stem cell transplantation include sickle cell disease, myelodysplastic syndromes, neuroblastoma, lymphoma, Ewing's sarcoma, desmoplastic small round cell tumor, and Hodgkin's disease. More recently, nonmyeloablative, or so-called "mini-transplant" techniques have been developed that require lower doses of pre-chemotherapy and radiation. This has allowed HSCT to be performed in elderly and other patients who may otherwise be considered too weak to tolerate conventional treatment regimens.

[0609] In one embodiment, the population of hematopoietic stem cells prepared by the methods of the invention are administered as part of an autologous stem cell transplantation procedure.

[0610] In another embodiment, the population of hematopoietic stem cells prepared by the methods of the present invention are administered as part of an allogeneic stem cell transplant procedure.

[0611] In one embodiment, the population of T cells prepared according to the methods of the invention are administered as part of an autologous T cell transplant procedure.

[0612] In another embodiment, the population of T cells prepared according to the methods of the invention are administered as part of an allogeneic T cell transplant procedure.

[0613] As used herein, the term "autologous stem cell transplantation method" refers to a method in which the starting cell population, which is subsequently transduced by the method of the present invention, is obtained from the same subject as the subject to whom the transduced cell population is administered. Autologous transplantation methods are advantageous because they avoid problems associated with immune incompatibility and can be utilized for subjects regardless of the availability of a genetically matched donor.

[0614] As used herein, the term "allogeneic stem cell transplantation method" refers to a method in which the starting cell population, which is subsequently transduced by the method of the present invention, is obtained from a subject different from the subject to whom the transduced cell population will be administered. Preferably, the donor is genetically compatible with the subject to whom the cells will be administered, to minimize the risk of immune incompatibility.

[0615] A suitable dose of the transduced cell population is, for example, one that is therapeutically and / or prophylactically effective. The dose to be administered may vary depending on the subject and condition being treated, and can be readily determined by one of skill in the art.

[0616] Hematopoietic progenitor cells provide short-term engraftment. Thus, gene therapy by administration of transduced hematopoietic progenitor cells will provide a non-permanent effect in the subject. For example, the effect may be limited to 1-6 months after administration of the transduced hematopoietic progenitor cells.

[0617] Such hematopoietic progenitor cell gene therapy may be suitable for the treatment of acquired diseases, e.g., cancer, where time-restricted expression of the (potentially toxic) anticancer nucleotide of interest may be sufficient to eradicate the disease.

[0618] The present invention may be useful in the treatment of disorders listed in WO1998 / 005635, a partial list of which is provided here for ease of reference: cancer, inflammation or inflammatory diseases, dermatological disorders, fever, cardiovascular effects, bleeding, coagulation and acute phase response, cachexia, anorexia, acute infections, HIV infection, shock states, graft versus host reaction, autoimmune diseases, reperfusion injury, meningitis, migraine and aspirin-dependent antithrombosis; tumour growth, invasion and spread, angiogenesis, metastasis, malignancy, ascites and malignant pleural effusion; cerebral ischemia, ischemic heart disease, osteoarthritis, rheumatoid arthritis. rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis or endosclerosis.

[0619] Additionally, or alternatively, the present invention may be useful in the treatment of disorders listed in WO1998 / 007859. For ease of reference, a partial list is provided here: cytokine and cell proliferation / differentiation activity; immunosuppressant or immunostimulatory activity (e.g., for treating immune deficiencies including human immunodeficiency virus infection; regulating lymphocyte proliferation; treating cancer and many autoimmune diseases, and preventing transplant rejection or inducing tumor immunity); regulating hematopoiesis, e.g., treating myeloid or lymphoid diseases; promoting the growth of bone, cartilage, tendon, ligament and nerve tissue, e.g., for wound healing, burns, ulcers and the treatment of periodontal disease and neurodegeneration; inhibiting or activating follicle-stimulating hormone (regulating fertility); chemotactic / chemokinetic activity (e.g., to recruit specific cell types to sites of injury or infection); hemostatic and thrombolytic activity (e.g., to treat hemophilia and stroke); anti-inflammatory activity (e.g., to treat septic shock or Crohn's disease); as an antimicrobial agent; e.g., as a metabolism or behavior modulator; as an analgesic; treating certain deficiency disorders; in human or veterinary medicine, e.g., in the treatment of psoriasis.

[0620] Additionally or alternatively, the present invention may be useful in the treatment of disorders listed in WO1998 / 009985, a partial list of which is provided here for ease of reference: macrophage inhibitory activity and / or T cell inhibitory activity, and thus anti-inflammatory activity; anti-immune activity, i.e. an inhibitory effect on cellular and / or humoral immune responses, including responses unrelated to inflammation; inhibition of the adhesion capacity of macrophages and T cells to extracellular matrix components and fibronectin, and upregulated receptor expression in T cells; inflammation associated with arthritis, including rheumatoid arthritis, hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary Inflammation associated with diseases, inflammation associated with peptic ulcer, ulcerative colitis and other gastrointestinal diseases, liver fibrosis, liver cirrhosis or other liver diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urinary diseases, otitis or other otorhinolaryngological diseases, dermatitis or other skin diseases, periodontal disease or other dental diseases, orchitis or epididymitis, infertility, testicular trauma or other immune-related testicular diseases, placental insufficiency, placental insufficiency, habitual abortion, eclampsia, pre-eclampsia 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 edema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, degenerative fundus diseasesimmune and inflammatory components of ocular diseases, inflammatory components of ocular injuries, ocular inflammation caused by infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring, e.g., following glaucoma filtration surgery, immune and / or inflammatory responses to intraocular implants, and other immune and inflammation related ocular diseases, inflammation associated with autoimmune diseases or conditions or disorders in which immune and / or inflammatory suppression would be beneficial in the therapy of the central nervous system (CNS) or any other organ, Parkinson's disease, complications and / or side effects from the treatment of Parkinson's disease, AIDS-related dementia complex HIV-associated encephalopathy, Devic's disease, Didenham's chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, Stokes' inflammatory component, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillain-Barré syndrome, Didenham's chorea, myasthenia gravis, pseudotumor cerebri, Down's syndrome group, Huntington's disease, amyotrophic lateral sclerosis, the inflammatory component of CNS compression or CNS trauma or CNS infection, the inflammatory component of muscle atrophy and muscular dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous system, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplant 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 inhibiting unwanted immune responses and inflammation including inflammation associated with AIDS, to suppress or inhibit humoral and / or cellular immune responses, to treat or ameliorate monocytic or leukoproliferative disorders such as leukemia by reducing the amount of monocytes or lymphocytes, to prevent and / or treat graft rejection in the case of transplantation of natural or artificial cells, tissues and organs such as corneas, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissues, etc.

[0621] Additionally, or alternatively, the present invention may be useful in the treatment of β-thalassemia, chronic granulomatous disease, metachromatic leukodystrophy, mucopolysaccharidoses and other lysosomal storage diseases.

[0622] kit In one aspect, the invention provides a kit comprising one or more inhibitors according to the invention, and / or a cell population of the invention.

[0623] In another aspect, the present invention provides a kit comprising one or more inhibitors according to the present invention, one or more nucleotide sequences encoding a gene editing mechanism and means for selecting hematopoietic stem cells.

[0624] The one or more inhibitors and / or cell populations according to the present invention may be provided in a suitable container.

[0625] Preferably, the kit may include a MAPK inhibitor. Preferably, the kit may include an IL-1 inhibitor. Preferably, the kit may include an NF-κB inhibitor. Preferably, the kit may include a MAPK inhibitor and an IL-1 inhibitor. Preferably, the kit may include a MAPK inhibitor and an NF-κB inhibitor. Preferably, the kit may include an IL-1 inhibitor and an NF-κB inhibitor. Preferably, the kit may include a MAPK inhibitor, an IL-1 inhibitor and an NF-κB inhibitor.

[0626] Suitably, the kit may comprise a nucleic acid sequence encoding at least one adenoviral protein.

[0627] The kit may also include instructions for use.

[0628] Treatment Method In a further aspect, the present invention provides a method for producing a method for the treatment of a cancer (a) gene editing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells according to the methods of the invention; and (b) administering to the subject gene-edited hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells. The present invention provides a method of gene therapy comprising:

[0629] In some embodiments, the gene-edited cells are administered to a subject as part of an autologous or allogeneic stem cell transplant procedure.

[0630] In a further aspect, the present invention provides a method for producing a method for the treatment of a cancer (a) transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells according to the methods of the invention; and (b) administering to a subject the population of transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells. The present invention provides a method of gene therapy comprising:

[0631] In some embodiments, step (b) comprises administering the transduced cells to the subject as part of an autologous or allogeneic stem cell transplant procedure.

[0632] The method of gene therapy may be, for example, a method of treating a disease selected from the group consisting of mucopolysaccharidosis type I (MPS-1), chronic granulomatous disease, Fanconi anemia (FA), sickle cell disease, metachromatic leukodystrophy (MLD), globoid cell leukodystrophy (GLD), GM2 gangliosidosis, thalassemia and cancer.

[0633] The method of gene therapy may be, for example, a method of treating diseases caused by Rag-1 mutations, such as SCID, atypical SCID and Omenn's syndrome.

[0634] In one embodiment, the subject is a mammalian subject, preferably a human subject.

[0635] In a further aspect, the present invention provides a population of gene-edited hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells prepared according to the methods of the present invention.

[0636] In a further aspect, the present invention provides a population of transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells prepared according to the methods of the present invention.

[0637] In a further aspect, the present invention provides a pharmaceutical composition comprising a population of gene-edited hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells of the present invention.

[0638] In a further aspect, the present invention provides a pharmaceutical composition comprising a population of transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells of the present invention.

[0639] In a further aspect, the present invention provides a population of gene-edited hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells of the invention for use in therapy, preferably gene therapy.

[0640] In a further aspect, the present invention provides a population of transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells of the invention for use in therapy, preferably gene therapy.

[0641] In some embodiments, the population is administered as part of an autologous or allogeneic stem cell transplant procedure.

[0642] All references to treatment herein should be understood to include curative, palliative and prophylactic treatment, although in the context of the present invention reference to prevention more generally relates to prophylactic treatment. In one embodiment, mammalian, particularly human, treatment is preferred. Both human and veterinary treatment are within the scope of the present invention.

[0643] A person skilled in the art will understand that he can combine all features of the invention disclosed herein without departing from the scope of the invention disclosed.

[0644] Administration The inhibitors and / or cells for use in the present invention (particularly the populations of cells produced by the methods of the present invention) may be administered alone but will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human treatment.

[0645] dose A person skilled in the art can easily determine the appropriate dose of one of the agents (e.g., inhibitors and / or cells) of the present invention to be administered to a subject without undue experimentation.Generally, a physician will determine the actual dose that is most suitable for an individual patient, which will vary depending on a variety of factors, including the activity of the specific agent employed, its metabolic stability and duration of action, age, weight, health condition, sex, diet, mode and time of administration, excretion rate, drug combination, the severity of the specific pathology, and the individual being treated.Of course, there may be individual cases in which higher or lower doses are appropriate, and these are within the scope of the present invention.

[0646] subject "Subject" refers to either a human or non-human animal.

[0647] Examples of non-human animals include vertebrates, such as mammals, such as non-human primates (particularly higher primates), dogs, rodents (e.g., mice, rats or guinea pigs), pigs and cats. The non-human animals may be companion animals.

[0648] Preferably, the subject is a human.

[0649] Mutants, derivatives, homologues and fragments In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses the use of mutants, derivatives, analogs, homologs and fragments thereof.

[0650] In the context of the present invention, a variant of any given sequence is a sequence in which a particular sequence of residues (whether amino acid or nucleic acid residues) has been altered in such a way that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or mutation of at least one residue present in the native protein.

[0651] Variant sequences of the SEQ ID NOs listed herein may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the reference sequence of the SEQ ID NOs. Preferably, the variant sequence retains one or more functions of the reference sequence (i.e., is a functional variant).

[0652] The variant sequence may contain one or more conservative substitutions. Conservative amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and uncharged polar head group amino acids with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0653] Conservative substitutions can be made, for example, according to the following table: Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

[0654] TIFF2024544746000044.tif79143

[0655] The present invention also encompasses homologous substitution (both substitution and replacement are used herein to mean the exchange of an existing amino acid residue for another) variants, i.e., like-for-like substitutions, such as basic for basic, acidic for acidic, polar for polar, etc.

[0656] Unless otherwise expressly stated herein by reference to a specific, individual amino acid, amino acids may be substituted with conservative substitutions such as those listed below.

[0657] The aliphatic, non-polar amino acid can be a glycine, alanine, proline, isoleucine, leucine, or valine residue.

[0658] The aliphatic, polar uncharged amino may be a cysteine, serine, threonine, methionine, asparagine, or glutamine residue.

[0659] The aliphatic, polar charged amino acid may be an aspartic acid, glutamic acid, lysine or arginine residue.

[0660] The aromatic amino acid may be a histidine, phenylalanine, tryptophan or tyrosine residue.

[0661] Preferably, conservative substitutions are made between amino acids in the same row of the above table.

[0662] As used herein, "sequence identity" is determined by comparing the sequence of a reference amino acid sequence to a portion of another amino acid sequence that has been aligned to maximize overlap between the two sequences while minimizing sequence gaps (not including any overhanging sequences between the two sequences).

[0663] Homology comparisons can be performed by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology or identity between two or more sequences.

[0664] The percentage of homology can be calculated over contiguous sequences, i.e., by aligning one sequence with the other and directly comparing each amino acid in one sequence with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are only performed over a relatively short number of residues.

[0665] While this is a very simple and consistent method, it fails to take into account, for example, that in an otherwise identical pair of sequences, a single insertion or deletion of a nucleotide sequence may move the following codon out of alignment, resulting in a significant drop in the percent homology when a global alignment is performed. As a result, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions, without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment and attempting to maximize local homology.

[0666] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, such that for the same number of identical amino acids, sequence alignments with as few gaps as possible achieve a higher score than those with many gaps, reflecting a higher relatedness between the two sequences being compared. "Affine gap costs" are commonly used, which impose a relatively high cost for the presence of a gap and a smaller penalty for each residue in the gap. This is the most commonly used gap scoring system. High gap penalties naturally produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, when using such software for sequence comparisons, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0667] Thus, the calculation of the maximum percentage of homology first requires the creation of an optimal alignment, taking into account gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software capable of carrying out sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid-Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS comparison toolset. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pp. 7-58 to 7-60). However, for some applications it is preferred to use the GCG Bestfit program. Another tool called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8).

[0668] Although the final percent homology can be measured in terms of identity, the alignment process itself is usually not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​commonly used that assigns a score to each matched comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix, which is the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the published default values ​​or custom symbol comparisons if provided (see user manual for details). Depending on the application, it may be preferable to use the published default values ​​for the GCG package, or a default matrix such as BLOSUM62 for other software.

[0669] Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity The software typically does this as part of the sequence comparison and generates a numerical result.

[0670] The term "homology" can be equivalent to the term "identity."

[0671] Preferably, references to a sequence having a certain percent identity to any one of the SEQ ID NOs detailed herein refer to a sequence having the stated percent identity over the entire length of the indicated SEQ ID NO.

[0672] The disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise specified, nucleic acid sequences are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0673] Where a range of values ​​is disclosed, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range is specifically disclosed. Each smaller range between any stated value or intervening value and any other stated value or intervening value in a stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range included in the smaller range where either, both or both limits are specifically excluded in the stated range is also encompassed within the disclosure. Where a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the disclosure.

[0674] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0675] The terms "comprising," "comprises," and "comprising of," as used herein, are synonymous with "including," "includes," or "contains," and indicate inclusion or open-endedness and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising," "comprises," and "comprising of" also include the term "consisting of."

[0676] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0677] The present invention will now be further described by way of examples, which are intended to aid those skilled in the art in practicing the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0678] Example 1 Anakinra treatment during gene editing improves HSPC clonogenic potential We previously reported the induction of a human hematopoietic stem and progenitor cell (HSPC) DNA damage response (DDR)-dependent proinflammatory program and its downstream effects on edited HSPC function (Schiroli et al., 2019, Cell Stem Cell 24: 551-565). We hypothesized that DDR-dependent inflammation would also affect gene editing (GE) of HSPCs. To counteract the reported and / or potential effects of DDR-dependent inflammation, we employed anakinra, a receptor antagonist of IL-1 (Cavalli and Dinarello, 2018, Front. Pharmacol. 9: 1157). IL-1 has been reported to be an upstream mediator of DDR-dependent inflammation (Pietras, 2017, Blood 130: 1693-1698; and Gnani et al., 2019, Aging Cell 18: e12933).

[0679] We edited the adeno-associated virus site 1 (AAVS1) locus in human umbilical cord blood (CB) HSPCs as a model safe harbor for targeted transgene insertion (Lombardo, A. et al., 2011, Nat. Methods 8: 861-869) by electroporating CRISPR / SpCas9 ribonucleoproteins (RNPs) with highly specific chemically modified guide RNAs (gRNAs) (Schiroli et al., 2019, Cell Stem Cell 24: 551-565). In addition, anakinra was added immediately after transfection of the GFP-expressing repair template AAV6 (hereafter, HS / AAV6 for gene-edited cells and HSPCs edited in the presence of anakinra, HS / AAV6+ANAK for anakinra-edited cells). As a negative control, we used RNPs with guide RNAs with no predicted activity in the human genome in the presence (-DSB+ANAK) or absence (-DSB) of anakinra (Figure 1A). First, we confirmed that anakinra treatment maintained the efficiency of GE across all HSPC subpopulations, from more committed to less differentiated subfractions, as demonstrated by the same percentage of HDR edited alleles in anakinra-treated edited cells compared to cells treated with our standard protocol (Figure 1B, C). Second, we measured the expression of the DDR downstream effector CDKN1A (hereafter referred to as p21) as a surrogate marker of DDR activation, as previously reported (Schiroli et al., 2019, Cell Stem Cell 24: 551-565). p21 levels were elevated in GE compared to negative controls, independently of anakinra treatment (Figure 1D), and this effect was maintained at 24 and 96 hours after treatment. Furthermore, we found that gene-edited HSPCs generated significantly fewer colonies in methylcellulose assays (Figure 1E), whereas anakinra treatment significantly increased the clonogenic potential of HSPCs, which was maintained after GE (Figure 1E). None of the treatments altered the culture composition over time (Figure 1F).

[0680] Overall, these data indicate that anakinra treatment during gene editing did not affect the repair capacity and p21 levels of HDR-induced HSPCs, but strongly improved the in vitro clonogenic potential of HSPCs.

[0681] Example 2 Anakinra suppresses the proinflammatory program resulting from GE of HSPCs Next, to evaluate whether anakinra could affect transcriptional changes occurring during GE and to investigate possible early and late effects of anakinra on edited HSPCs, we performed whole-transcriptome analysis of CB-derived HSPCs 24 and 96 h after AAVS locus editing. Editing was examined in the presence or absence of anakinra in edited HSPCs and the respective negative controls. To identify pathways regulated by anakinra, we performed GSEA on gene lists ranked based on log2FC. We found negative and significantly higher normalized enrichment scores (NES) for inflammatory / TNFα-dependent pathways (TNF-α signaling via nuclear factor kappa B [NFκB]; IL2-STAT5 signaling; IL-6 / JAK / STAT3 signaling) and interferon response in cells edited in the presence of anakinra compared to untreated controls (Figure 2A,B). As a result, we observed a significant upregulation of key inflammatory genes in the IL-1 signaling pathway in edited HSPCs, which was downregulated 96 h after anakinra treatment (Figure 2C).

[0682] Taken together, these studies suggest that the induction of a proinflammatory program at early and late stages after gene editing is downregulated at early and late stages by anakinra treatment.

[0683] Example 3 Blockade of the IL-1 signaling pathway is a method to maintain long-term reconstitution of HDR-edited cells in vivo To examine the repopulation potential of HSPCs edited in the presence or absence of anakinra, we transplanted matched saturating cell doses into NOD Prkdcscid Il2rg- / - (NSG) mice. Additionally, as a positive control, cells edited in the presence of p53 inhibition (co-electroporated with mRNA encoding a dominant-negative p53 truncated form (GSE56) as previously described (Schiroli et al., 2019, Cell Stem Cell 24: 551-565; and Ferrari et al., 2020, Nat Biotechnol 38: 1298-1308)) were used alone or in combination with anakinra. Comparable human engraftment rates were observed between treatment regimens, reaching a plateau of 35-40% of circulating cells (Figure 3A). Similar engraftment patterns were also seen long-term after hematopoietic organ reconstitution (Figure 3C). Considering the repopulation of HDR-edited HSPCs in humans, we found that treating cells with anakinra resulted in increased expression of GFP- + A high and stable percentage of cells was observed (Figure 3B). + The percentages of cells, sorted progenitors and lineages were consistent with the levels observed in blood, and HDR-edited cells treated with anakinra showed improved long-term reconstitution compared to standard protocol edited cells (Figure 3D,E).

[0684] In addition, human CD34 cells harvested from the bone marrow of primary recipients + When cells were re-administered in semi-solid medium, we found that anakinra-treated edited HSPCs exhibited enhanced clonogenic potential compared to standard protocol-treated cells (Figure 3F), which was consistent with reduced pro-inflammatory cytokine expression (Figure 3G).

[0685] To assess the clonal composition of host repopulation with anakinra-edited HSPCs, we embedded a 22 base pair (bp) degenerate heritable "barcode" sequence (BAR) into the repair template downstream of a green fluorescent protein (GFP) reporter cassette. We then cloned HSPCs of high equivalent complexity (7.5 × 10, respectively) into the repair template. 5 and 5.9 x 105 We generated plasmid libraries and AAV6 pools with near-uniform representation of 10 unique BARs and degenerate consensus sequences (Ferrari et al., 2020, Nat Biotechnol 38: 1298-1308). Anakinra-treated edited HSPCs were found to exhibit higher polyclonal reconstitution compared to standard gene-edited cells, with an increased number of dominant BARs compared to the respective controls (Figure 3H). Polyclonal reconstitution after anakinra treatment was also confirmed by analyzing PBMCs in a saturating dose experiment (Figure 3G).

[0686] Overall, these data suggest that in the presence of anakinra, HDR-edited HSPCs exhibit higher long-term polyclonal reconstitution, which is associated with reduced proinflammatory cytokines.

[0687] Example 4 Anakinra reduces nuclear translocation of NF-KB during GE To study the molecular mechanisms underlying IL-1-mediated inflammation, we measured the localization of nuclear factor kappa beta (NF-κB) as a surrogate marker of its activation, as previously reported. During the gene editing procedure, edited cells were treated with anakinra, and further controls were used to distinguish the effect of anakinra. Single electroporation of cells (hereafter, UT electro sample), transduction of cells after electroporation (hereafter, UT electro+AAV6 sample), and standard gene editing procedure (HS / AAV6 sample) were performed in the presence or absence of anakinra. Compared with the negative controls (UT electro and UT electro+AAV6), we found that the nuclear translocation of NF-κB upon gene editing was increased and decreased upon anakinra treatment (Figure 4A-B), suggesting that the IL-1 signaling pathway is upstream of the activation of NF-κB during GE. Because anakinra treatment is transient, NF-κB activation was no longer observed 96 h after GE.

[0688] Example 5 NF-κB inhibition improves long-term reconstitution of HSPCs and synergizes with p53 inhibition To more broadly inhibit the NF-κB pathway and better improve the function of edited HSPCs, we employed the IKK-2 inhibitor (SC514), a suppressor of the NF-κB-dependent inflammatory transcriptional program (Kishore, N. et al., 2003, Journal of Biological Chemistry 278: 32861-32871; and Roman-Blas, JA, & Jimenez, SA, 2006, Osteoarthritis Cartilage 14: 839-848). Cells were treated with SC514 during GE of the AAVS1 locus, p53 inhibition was performed alone or in combination with the NF-κB inhibitor, and cells were harvested 24 and 96 hours after GE (Figure 5A). Comparing HSPCs edited in the presence or absence of NF-κB inhibition, we found no adverse effect on genome editing efficiency for the HDR-edited allele. On the contrary, we found that GSE56 tended to increase the percentage of HDR editing, and this effect was maintained upon NF-κB inhibition (Figure 5B). Furthermore, because NF-κB is an upstream mediator of inflammatory programs, we observed a decrease in the expression levels of several upregulated DDR-associated cytokines (e.g., IL-6, IL-8, and CCL2) in edited HSPCs treated with SC514. Furthermore, we also observed a downregulation of cytokines in the presence of GSE56, which was further exacerbated by simultaneous (i.e., combined) treatment of GSE56 and SC514 (Figure 5C). Colony formation potential was similarly increased by editing treatment in the presence of SC514 or GSE56 compared to HSPCs edited with the standard protocol, and the GSE56 / SC514 combination tended to produce more colonies, although there was no detectable difference in the erythroid to myeloid ratio (Figure 5D). To test the repopulation potential of HSPCs edited in the presence or absence of SC514 and GSE56, we transplanted the limiting cell dose into NSG mice and confirmed that the addition of GSE56 enabled four-fold increased engraftment compared to the standard protocol, and in combination with SC514, this increase was further enhanced, reaching nearly 40% of human engraftment (Figure 5E). Similar engraftment patterns were observed long-term after reconstitution in hematopoietic organs (Figure 5F).To further examine the long-term function of edited HSPCs, we transfected them with human CD34 from the bone marrow of primary recipients. + We performed colony formation assays by purifying the cells (Figure 5G). We showed that the clonogenic potential of cells was slightly increased upon SC514 treatment, which was further increased when combined with p53 inhibition. Furthermore, combined NF-κB and p53 inhibition significantly increased the fitness of edited HSPCs, suggesting a synergistic effect. Finally, we performed clonal tracking of HDR-edited HSPCs by a barcode-based strategy, and found that NF-κB inhibition improved polyclonal reconstitution, while combined with GSE56 maintained the self-renewal and pluripotency of individual edited HSPCs.

[0689] Example 6 Edited HSPCs pretreated with p38 inhibitors exhibit increased clonogenic potential and reduced ROS levels To improve the culture conditions and ultimately obtain more functional edited HSPCs, we considered inhibiting p38-MAPK signaling using SB203580, a highly specific ATP-competitive kinase activity inhibitor that does not affect protein phosphorylation. Specifically, we decided to treat HSPCs during the first 2 days of culture, since standard gene editing protocols require gene editing to be performed on day 3 after thawing. In detail, HSPCs were treated with DMSO or p38i (4 μM) on days 1 and 2. On day 3, HSPCs were electroporated with HS RNPs alone or with the addition of AAV6 GFP-expressing donor template, as required for standard gene editing protocols (Schiroli et al., 2019, Cell Stem Cell 24: 551-565) (Figure 6A).

[0690] To assess HSPC function, we next tested their clonogenic potential and found that under both HS RNP and HS / AAV6 conditions, there was an increase in the number of colonies following p38 inhibitor treatment. Surprisingly, the increase in colony number was mostly due to an increase in the number of mixed colonies, which were more undifferentiated and multipotent progenitors of HSCs (Carow, CE, Hangoc, G. and Broxmeyer, H. E, 1993, Blood, 81: 942-949) (Figure 6B). Interestingly, two doses of 4 μM p38 inhibitor on days 1 and 2 post-thawing were able to reduce the accumulation of ROS and mitochondrial superoxide in HSPCs during editing (Figure 6C).

[0691] Example 7 Edited HSPCs pretreated with p38 inhibitors show high engraftment and number of edited clones after transplantation in NSG mice The increase in mixed colonies seen when HSPCs were edited upon p38 inhibition suggested that this treatment could maintain the fitness of more undifferentiated HSCs. Therefore, we tested the in vivo function of HSPCs upon single DSB (HS) or full editing (HS / AAV6) in the presence or absence of p38 inhibition. HSPCs were treated with DMSO (as control) or 4 μM p38i on days 1 and 2, and CB-derived CD34 cells treated with HS RNPs alone or in combination with AAV6 donor templates were cultured. + HSPCs were transplanted into NOD Prkdcscid Il2rg (NSG) mice (Figure 7A). Serological analysis was performed at different time points (6–15 weeks) after transplantation to detect human CD45 in peripheral blood. + (hCD45 + The repopulating ability of HSPCs was tracked by measuring the percentage of hCD45 cells in mice transplanted with HSPCs that had only experienced DSB (HS conditions) pretreated with p38 inhibitors. +The percentage of cells effectively increased until 12 weeks, but stabilized at 15 weeks, with engraftment returning to similar levels to the DMSO control. Instead, engraftment rates of HS / AAV6+p38i-treated HSPCs were higher from the start compared to the DMSO control group (Figure 7B). Data obtained in bone marrow (BM) at the time of euthanasia (15w) mirrored the findings in PB. Engraftment in HS conditions was comparable between the two treatments, but p38 inhibition improved engraftment of HS / AAV6 (Figure 7C). Given the encouraging results regarding engraftment and the improved function of edited HSPCs upon p38 inhibitor treatment, we asked whether p38 inhibition could affect the number of edited HSPC clones. Indeed, of mice transplanted with DMSO- or p38 inhibitor-edited HSPCs, GFP-GFP was not detected in the BM at 15w. + Mice with detectable (>0.1%) cell engraftment were selected for BAR-seq analysis (Ferrari et al., 2020, Nat Biotechnol 38: 1298-1308). Five of six mice in the HS / AAV6+DMSO group and five of six mice in the HS / AAV6+p38i 4μM group were included. Interestingly, the number of dominant BARs was significantly increased upon p38 inhibition, suggesting that polyclonal reconstitution of HDR-edited cells was promoted upon p38i treatment (Figure 7D).

[0692] Example 8. p38 Inhibition Increases HSPC Function and Reduces the Percentage of Senescent Cells Long-Term After Transplantation At the time of euthanasia (15 weeks), CD34 was isolated from bone marrow. + The cells were purified and tested for clonogenic potential in a methylcellulose assay (Figure 7E). Interestingly, we observed that BM-derived HS and HS / AAV6 HSPCs treated with p38 inhibitors formed a higher number of colonies, even though the difference was more pronounced in the HS / AAV6 group. As previously mentioned, this increase in colony number was due to an increase in mixed colonies in both conditions upon p38i treatment (Figure 7F).

[0693] Consistent with these results, we also observed a reduction in the presence of senescent HSPCs when pretreated with p38 inhibitors, confirming that p38 inhibition better preserves the function of edited HSPCs ( Figure 7F ).

[0694] Example 9 Inhibition of Different MAPKs Increases HDR in Different Subpopulations and Improves the Clonogenic Potential of HSPCs Given the promising results obtained with p38 inhibition, we suspected that activation of other mitogen-activated protein MAP kinases (MAPKs) may contribute to the ex vivo expansion and loss of HSPC function following genome editing. Indeed, MAPK family members are evolutionarily conserved and are involved in the control of physiological cellular processes including cell proliferation, survival, differentiation, apoptosis and tumorigenesis. In mammalian cells, three distinct subgroups within the MAPK family have been reported: classical MAPKs (also known as ERKs), C-Jun N-terminal kinase / stress-activated protein kinases (JNKs / SAPKs), and p38 kinases (Wei, Z. and Liu, HT, 2002, Cell Research. Science Press 12: 9-18; and Cicenas et al., 2018, Cancers 10: 63). In detail, HSPCs were treated with DMSO, p38 (4 μM), Erk (4 μM) or Jnk (2 μM) inhibitors on days 1 and 2. On day 3, HSPCs were electroporated with HS RNPs alone or in addition to the AAV6 GFP-expressing donor template required for standard gene editing protocols (Figure 8A). We examined GE efficiency in subpopulations of HSPCs, from more committed to less differentiated subfractions, and found increased GFP levels upon inhibition of Erk and Jnk (Figure 8B).

[0695] Furthermore, we tested the clonogenic potential of HSPCs to assess functionality, and found that an increase in mixed colonies was observed after all inhibitor treatments in both HS RNP and HS / AAV6 conditions (Figure 8C).

[0696] Example 10. p38 inhibitor treatment reduces the percentage of mitochondrial superoxide and senescence. Because p38 inhibition improved HSPC function during gene editing, we sought to test it in other clinically relevant cell types for gene and cell therapy. + Starting with T cells, we tested two concentrations of p38 inhibitors. Specifically, T cells were treated with 4 μM or 10 μM p38i on days 1 and 2 and edited on day 3 (Figure 9A). Mitochondrial superoxide levels were measured 24 hours after editing and found to be elevated upon gene editing compared to the unedited control (UT), but importantly, both doses of p38i reduced the levels (Figure 9B).

[0697] Furthermore, increased cell proliferation and decreased senescence of edited cells were observed upon p38 treatment (data not shown).

[0698] Example 11 Gene editing leads to activation of a senescence program that impairs HSPC function for a long period of time To investigate unintended effects of gene editing methods on human HSC function, we used CB-derived CD34 +We employed our optimized protocol, consisting of culturing the cells in early-active cytokine and stem-preserving activity medium for 3 days (Schiroli, G. et al., 2019, Cell Stem Cell 24: 551-565). We then performed gene editing of the safe harbor AAVS1 locus using highly purified clinical-grade CRISPR-Cas9 ribonucleoprotein complexes and AAV6 delivering a GFP-expressing DNA repair template for HDR (Figure 10A). Cells were harvested 24 hours (h) and 96 hours (h) after gene editing, and the edited cells (HS / AAV6) showed an increase in the senescence-associated marker p16 compared to cells that had received electroporation alone (untreated electrp, UT EL) (Figure 10B). In addition, we also performed a side-by-side comparison of all reagents involved in the GE method and measured the accumulation of the lysosomal enzyme senescence-associated β-galactosidase (SA-β-gal), a senescence marker, using increasing doses of AAV6 as the DNA donor template for HDR. We reported that transduction with increasing doses of AAV6 donor template elicited a senescence-like phenotype at 24 h post-GE compared to control cells only subjected to electroporation, but this response was transient and disappeared at 94 h post-editing. Conversely, GE resulted in a higher level of senescence induction compared to electroporation and AAV6 transduction alone, and such a response was maintained over time, especially in cells that underwent HDR (GFP positive) (Figure 10C). Furthermore, we reported that increasing doses of AAV6 transduction impaired the clonogenic potential of HSPCs, but this impairment was more evident with the full GE method (Figure 10D). Interestingly, HDR-edited cells were also observed to exhibit senescence features in clonogenesis, and indeed, GFP-positive cells were also observed to exhibit senescence features in clonogenesis. + In colonies derived from cells, GFP - We found elevated levels of SA-β-gal compared to cell-derived colonies (Figure S10E). Consistent with this, when edited cells were transplanted into NSG mice, we found that long-term hematopoietic reconstitution was impaired compared to UT EL controls, particularly in cells that received GE at the highest AAV6 dose (Figure S10F,G,H).

[0699] Next, we examined the senescence burden in human grafts grown from transplanted edited cells into NOD Prkdcscid Il2rg- / - (NSG) mice, and found that all human CD45 + Cells (Figure 10I) and BM-derived CD34 + In cells (Figure 1J), unedited GFP - We report increased induction of senescence in HDR-edited cells compared to cells (light grey) and cells that underwent electroporation (black) or AAV6 transduction (dark grey) alone. Overall, these data indicate that GE in HSPCs culminates in the establishment of a senescence program that impacts HSPC function in the long term.

[0700] Example 12 Transient DDR inhibition prevents accumulation of senescence in HDR-edited HSPCs To assess the functional role of cellular senescence in edited HSPCs, we transiently inhibited the DDR pathway, which acts on the DDR apical kinase ATM during ex vivo editing, using a p53 inhibitor (GSE56) (Figure S1A). We reported that transient inhibition of ATM within 15 min after GE did not impair the repair capacity of HSPCs by HDR (Figure S1B). Indeed, the most undifferentiated progenitors (CD34 + CD133 + CD90 + cells) and more committed progenitor cells (CD34 + CD133 - , CD34 + CD133 + ) in + The percentage of cells was similar in HSPCs treated with ATM inhibitor (ATMi) compared to their untreated counterparts (Figure 11C).

[0701] Activation of inflammatory pathways is a key feature of senescent cells. Since ATM kinase has been reported to directly activate NF-κB, a key transcription factor that plays a key role in the transcriptional activation of inflammatory cytokines (Fang, L. et al. Nucleic Acids Res 2014. 42: 8416-8432), we hypothesized that ATM may also be involved in GE-associated inflammation. By performing gene expression analysis of several key inflammatory cytokines previously reported to be regulated by DDR, we found that edited HSPCs showed upregulation of IL1A, IL6 cytokines reported 24 hours after GE, and ATM inhibition suppressed this induction (Figure 11D). Furthermore, we tested the functional properties of the cells and found that edited HSPCs showed a decrease in clonogenic potential over time compared to the negative control (UT EL DMSO), which was improved by ATM inhibition. However, we reported that even in cells that were only electroporated, clonogenic potential was slightly improved upon ATM inhibition (Figure 11E). Furthermore, a trend towards increased engraftment was observed early in transplantation in vivo (Figure 11F), confirming that ATMi improves the repopulation capacity of edited HSPCs, suggesting that ATMi exerts beneficial effects mainly on HSPC progenitors involved in short-term repopulation. Indeed, we did not report any difference in engraftment of BM-derived HSPCs in the presence or absence of ATM inhibition (Figure 11G). As we previously reported that p53 inhibition is a useful strategy to increase HSPC engraftment and clonality (Schiroli, G. et al., 2019, Cell Stem Cell 24: 551-565, Ferrari, S. et al., 2020, Nat Biotech 38(11):1298-1308), we reasoned that administering GSE56 at the time of GE might also help to alleviate the establishment of GE-mediated cellular senescence. p53 inhibition improved the engraftment of HDR-edited BM-derived CD34 +We found that the accumulation of the senescence marker SA-β-gal in HSPCs was suppressed (Figure S1H). Finally, HSPCs harvested from bone marrow of mice transplanted with edited cells (HS / AAV6) showed elevated p21, p16, and IL8 mRNA levels, which were mitigated by treatment with GSE56 at the time of ex vivo editing (Figures S1I, S1J, S1K), consistent with improved clonogenic potential of HSPCs upon p53 inhibition when BM-derived cells were re-fed in semi-solid medium (Figure S1L).

[0702] Together, these results support the idea that gene editing can inadvertently induce a DDR-dependent senescence program in transplanted HSPCs.

[0703] Example 13 Anakinra treatment during gene editing improves HSPC clonogenic potential Because p53 inhibition may exacerbate the genotoxicity risks of gene editing, we turned to modulating downstream GE-associated inflammation.To counteract the previously reported induction of proinflammatory and senescence-associated programs in human hematopoietic stem and progenitor cells (HSPCs) and their downstream effects on edited HSPC function (Schiroli et al., 2019, Cell Stem Cell 24: 551-565), we used anakinra, an IL1 receptor antagonist (Cavalli and Dinarello, 2018, Front. Pharmacol. 9: 1157), which has been widely reported to be an upstream mediator of HSC exhaustion, DDR-dependent inflammation, and a key component of the senescence-associated inflammatory program (Pietras, 2017, Blood 130: 1693-1698; and Gnani et al., 2019, Aging Cell 18: e12933). Indeed, we reasoned that inhibiting the IL1 signaling pathway with recombinant IL1 receptor antagonist anakinra within 15 min after GE may help maintain long-term functionality of corrected HSPCs. We edited the adeno-associated virus site 1 (AAVS1) locus (Lombardo, A. et al., 2011, Nat. Methods 8: 861-869) as a model safe harbor for targeted transgene insertion in human umbilical cord blood (CB)-derived HSPCs by electroporating CRISPR / SpCas9 ribonucleoproteins (RNPs) and highly specific chemically modified guide RNAs (gRNAs) (Schiroli et al., 2019, Cell Stem Cell 24: 551-565). Furthermore, we added anakinra within 15 min after transfection of the GFP-expressing repair template AAV6 (hereafter referred to as HS / AAV6 for gene-edited cells and HS / AAV6+ANAK for edited HSPCs in the presence of anakinra). As a negative control, we employed an RNP with a guide RNA with no predicted activity in the human genome in the presence (-DSB+ANAK) or absence (-DSB) of anakinra (Figure 12A).First, we confirmed that anakinra treatment did not affect the efficiency of GE, as indicated by a comparable percentage of HDR-edited alleles, by comparing anakinra-treated edited cells with cells treated with the standard protocol (Figure 12B). Furthermore, no significant differences in GE efficiency were observed across all HSPC subpopulations, from more committed to less differentiated subfractions (Figure 12C). Next, we measured the expression of the DDR downstream effector CDKN1A (hereafter referred to as p21) as a surrogate marker of DDR activation, as previously reported (Schiroli et al., 2019, Cell Stem Cell 24: 551-565). p21 levels were upregulated upon GE compared to negative controls, independently of anakinra treatment (Figure 12D), and this effect was maintained for 24 and 96 hours after treatment. Furthermore, gene-edited HSPCs generated significantly fewer colonies in methylcellulose assays, indicating a significant increase in HSPC clonogenic potential upon anakinra treatment that was maintained after GE (Figure S12E), whereas no treatment varied culture composition over time (Figure S12F).

[0704] Overall, these data indicate that anakinra treatment during gene editing did not affect the repair capacity of HSPCs via HDR and p21 levels, but strongly improved ex-vivo HSPC clonogenic potential.

[0705] Example 14 Anakinra inhibits inflammation and senescence as a result of GE of HSPCs Next, to assess whether anakinra affects transcriptional changes occurring during GE and to examine possible early and late effects of anakinra on edited HSPCs, we performed whole-transcriptome analysis of CB-derived HSPCs 24 and 96 h after AAVS locus editing. Editing was tested in the presence or absence of anakinra in edited HSPCs and the respective negative controls. To identify pathways regulated by anakinra, GSEA was performed on gene lists ranked based on log2FC. In cells edited in the presence of anakinra, we observed negative and significantly higher normalized enrichment scores (NES) relative to non-treated cells 24 h after GE, primarily in inflammation / TNFα-dependent pathways (e.g., TNF-α signaling via nuclear factor kappa B [NFκB], IL2-STAT5 signaling, IL-6 / JAK / STAT3 signaling), interferon response, and oxidative stress gene categories (Figures 13A, 13B). Accordingly, we reported a significant upregulation of key inflammatory genes in the IL1 signaling pathway in edited HSPCs, which were downregulated 96 h after anakinra treatment (Figure S13C). Indeed, qPCR showed a significant induction of IL1A, CXCL8 (interleukin-8 [IL8]), and IL6 in response to GE, which was upregulated in anakinra-treated edited cells.

[0706] Together, these studies revealed that the induction of pro-inflammatory and senescence programs at both the early and late stages of gene editing was downregulated by anakinra treatment.

[0707] Example 15 Anakinra reduces GE-induced NF-KB nuclear translocation To study the molecular mechanisms underlying IL-1-mediated inflammation, we measured the localization of nuclear factor kappa beta (NF-κB) as a surrogate marker of its activation. During the gene editing procedure, edited cells were treated with anakinra, and controls were added to distinguish the effect of anakinra. Single electroporation of cells (hereafter, UT electro sample), AAV6 transduction of cells after electroporation (hereafter, UT electro+AAV6 sample), and standard gene editing procedure (HS / AAV6 sample) were performed in the presence or absence of anakinra. Compared to the negative controls (UT electro and UT electro+AAV6), we found that the nuclear translocation of NF-κB increased upon gene editing and decreased upon anakinra treatment (Figures 14A, 14B). This suggests that the IL-1 signaling pathway is upstream of the activation of NF-κB during GE. Because anakinra treatment was transient, NF-κB activation 96 hours after GE was not reported. Together, these studies highlight that IL1 acts upstream of NF-κB to regulate the establishment of GE-associated inflammation and senescence.

[0708] Example 16 Blockade of the IL1 signaling pathway is a method to preserve long-term reconstitution of HDR-edited cells in vivo To examine the potential for repopulation of HSPCs edited in the presence or absence of anakinra, we transplanted a saturating dose of matching GE cells into NSG mice. Comparable human engraftment was seen independent of anakinra treatment, reaching a plateau of 35-40% of circulating cells (Figure 15A), but HDR-edited cells edited in the presence of anakinra had reproducible higher and more stable levels when compared to standard controls in peripheral blood (PB) over time (Figure 15B). Similar engraftment patterns and GFP expression were observed in human grafts after bone marrow reconstitution. + HDR-edited cells were seen over time (Figures 15C, 15D). GFP in BM-derived HSPCs, B cells, and myeloid cell lineages at endpoints +The percentage of cells consistent with levels seen in blood and BM, with HDR-edited cells treated with anakinra showing improved long-term reconstitution compared to cells edited with the standard protocol (Figure 15E). Furthermore, human CD34 + When the cells were re-administered in semi-solid medium, we found that anakinra-treated edited HSPCs exhibited higher clonogenic potential compared to cells treated with the standard protocol (Figure 15F). The higher engraftment of HDR-edited cells treated with anakinra was consistent with the prolonged expression of inflammatory cytokines (Figures 15G, 15H) and the reduced percentage of senescent cells in human grafts (Figure 15I). To assess the clonal composition of host repopulation with anakinra-edited HSPCs, we embedded a 22-base pair (bp) degenerate heritable "barcode" sequence (BAR) into the repair template downstream of a green fluorescent protein (GFP) reporter cassette. We then generated 1000 HSPCs with high equivalent complexity (7.5 × 10, respectively). 5 and 5.9 x 10 5 We generated plasmid libraries and AAV6 pools with near-uniform representation of HDR-edited clones (unique BARs) and degenerate consensus sequences (Ferrari et al., 2020, Nat Biotechnol 38: 1298-1308). We found that edited HSPCs treated with anakinra showed higher polyclonal reconstitution compared to standard gene-edited cells, with an increased number of dominant BARs compared to the respective controls (Figure 15J). Polyclonal reconstitution of short-term and long-term HDR-edited clones in human grafts upon transplantation after anakinra treatment was also confirmed by analyzing PBMCs in a saturating dose experiment (Figure 15K).

[0709] Overall, these data reveal that in the presence of anakinra, HDR-edited HSPCs exhibited a greater degree of long-term polyclonal reconstitution associated with suppression of inflammatory cytokines and senescence markers.

[0710] Example 17 Anakinra treatment improves mPB-derived HSPC clonogenic potential without affecting HDR-mediated HSPC repair capacity To extend the data to a more clinically relevant HSPC source, we performed the same experiments reported for CB-derived HSPCs on mPB-derived HSPCs. We treated cells within 15 min after gene editing of the AAVS1 locus with anakinra (Figure 16A) and demonstrated that anakinra treatment does not affect HDR-mediated HSPC repair capacity (Figure 16B) or alter HSPC culture composition and differentiation (Figure 16C), but significantly improves the clonogenic potential of edited cells (Figure 16D). Furthermore, GFP- + HDR-edited cells showed high accumulation of the senescence marker p16 in early and late GE, which was partially alleviated by anakinra treatment (Figure S16E). Consistently, when matched low-dose edited cells were transplanted into NSG mice, comparable human engraftment was seen independent of anakinra treatment (Figure S16F), but higher levels of HDR-edited cells were edited in the presence of anakinra when compared to standard controls (Figure S16G), tending to alleviate the senescence burden in human grafts (Figure S16H).

[0711] Overall, these data suggest that anakinra suppresses the establishment of GE-associated senescence also in mPB-derived HSPCs and improves long-term hematopoietic reconstitution.

[0712] Example 18 Anakinra treatment improves mPB-derived HSPC clonogenic potential without affecting HDR-mediated HSPC repair capacity Utilizing alternative DNA donor templates for HDR distinct from AAV6 To dissect the role of senescence and inflammation with respect to GE, we performed AAVS1 editing in mPB HSPCs using an integrase-deficient lentiviral vector (IDLV) as a template for HDR.

[0713] To achieve higher efficiency of IDLV transduction, we employed the transduction enhancer CsH as previously reported (Petrillo, C. et al. 2018, Cell Stem Cell 23, 820-832.e9). Furthermore, to achieve higher levels of HDR, we performed AAVS1 editing in mPB HSPCs by combining CsH, GSE56 and E4orf6 / 7 adenoviral proteins (GSE56+E4orf6 / 7, hereafter referred to as COMBO for the optimized protocol with HDR enhancers) (Ferrari, S. et al., 2020, Nat Biotech 38(11):1298-1308). To identify optimal conditions for maximizing DNA donor availability during DSB repair, we tested different timings (12 or 24 hours prior to RNP electroporation) and one or two hits of IDLV delivery compared to those of AAV6 (see protocols used in Figures 17A, 17B, 17C). We reported a sustained increase in the DDR marker NBS1 when cells were edited in the presence of AAV6 donor templates compared to one or two hit IDLV templates using the optimized protocol for all treatments (Figure 17D). Notably, this was consistent with higher NF-κB activation in AAV6 GE compared to one hit IDLV GE at early and late stages of genetic manipulation (Figure 17E). To suppress the higher nuclear activation of AAV6 NF-κB compared to IDLV, CB-derived HSPCs electroporated with HDR enhancer (COMBO) were subjected to anti-inflammatory treatment 15 min after GE with anakinra or the NF-κB inhibitor SC-514 (Kishore, N. et al., 2003, Journal of Biological Chemistry 278: 32861-32871; and Roman-las, JA, & Jimenez, SA, 2006, Osteoarthritis Cartilage 14: 839-848).We reported that both anti-inflammatory treatments further reduced the nuclear localization of NF-κB compared to cells that underwent GE in the presence of GSE56 and E4orf6 / 7 alone (Figure S17F). The reduced NF-κB induction upon anakinra or SC514 treatment was associated with an increased clonogenic potential of the cells (Figure S17J) without compromising the HDR-mediated HSPC repair capacity (Figure S17H), without overt apoptosis (Figure S17I) and without perturbations in culture composition (Figure S17G).

[0714] Overall, these findings reveal that GE with IDLV donor templates also led to induction of DDR and NF-κB in mPB-derived HSPCs, but with higher transduction efficiency but lower induction compared to AAV6 treatment.Furthermore, performing GE with AAV6 in the presence of HDR enhancers could attenuate NF-κB induction by transient inhibition of inflammatory pathways, leading to increased clonogenic potential of edited HSPCs.

[0715] Example 19 NF-κB inhibition improves long-term reconstitution of HSPCs and synergizes with p53 inhibition To more broadly inhibit the NF-κB pathway and better improve the function of edited HSPCs, we employed the IKK-2 inhibitor (SC514) mentioned above, which is a suppressor of the NF-κB-dependent inflammatory transcriptional program (Kishore, N. et al., 2003, Journal of Biological Chemistry 278: 32861-32871; and Roman-Blas, JA, & Jimenez, SA, 2006, Osteoarthritis Cartilage 14: 839-848). Within 15 min after GE of the AAVS1 locus in CB-derived HSPCs, cells were treated with SC514, p53 inhibition was performed alone or in combination with an NF-κB inhibitor, and cells were harvested at 24 and 96 h after GE (Figure 18A). No differences in HDR-edited alleles were observed when comparing HSPCs edited in the presence or absence of NF-κB inhibition. On the contrary, we found that GSE56 tended to increase the percentage of HDR editing, and this effect was maintained upon NF-κB inhibition (Figure 18B). Furthermore, because NF-κB is an upstream mediator of inflammation, we observed a decrease in the expression levels of several upregulated DDR-related cytokines (e.g., IL-6, IL-8, and CCL2) in edited HSPCs treated with SC514. Furthermore, we also observed a downregulation of cytokines in the presence of GSE56, which was further exacerbated by co-treatment of GSE56 and SC514 (Figure 18C). Colony formation ability was similarly increased by editing treatment in the presence of SC514 or GSE56 compared to HSPCs edited by the standard protocol, and the GSE56 / SC514 combination tended to produce more colonies, although there was no detectable difference in the erythroid to myeloid ratio (Figure 18D).

[0716] In addition, NF-kB inhibition experiments were performed on mPB-derived HSPCs, and GFP +While there were no differences in cells, particularly in the most undifferentiated subpopulations (Figure 18E), we observed improved clonogenic potential of edited cells undergoing NF-kB inhibition over time compared to untreated controls (Figure 18F), which correlated with a reduced establishment of senescence in HDR-edited cells (Figure 18G). Together, these data suggest that transient inhibition of NF-kB can increase the clonogenic potential of edited HSPCs by reducing the accumulation of senescence markers, which is further improved by the addition of GSE56.

[0717] Example 20 NF-κB inhibition during GE reduces the percentage of HDR-edited senescent cells in human grafts To test the repopulation potential of HSPCs edited in the presence or absence of SC514 and GSE56, we transplanted the matching limiting cell dose into NSG mice and confirmed that the addition of GSE56 resulted in four-fold increased engraftment over the standard protocol, and this increase was further enhanced in combination with SC514, reaching nearly 40% human engraftment (Figure 19A). A similar pattern of engraftment was observed long-term after hematopoietic reconstitution (Figure 19B). To further test the long-term function of edited HSPCs, we isolated human CD34 HSPCs from the bone marrow of primary recipients. + We performed colony formation assays by purifying the cells. We showed a slight trend toward increased clonogenic potential of cells upon SC514 treatment, which was further increased upon p53 inhibition (Figure 19C). Furthermore, we reported that combining NF-κB inhibition with p53 inhibition significantly increased the fitness of edited HSPCs, suggesting a possible synergistic effect.

[0718] Finally, by performing clonal tracking of HDR-edited HSPCs by a barcode-based strategy (Figure 19D), as well as clonal tracking of alleles with unique indels in human explants (Figure 19E), we found that NF-kB inhibition improved polyclonal reconstitution and maintained the self-renewal and pluripotency of individual edited HSPCs in combination with GSE56....

Claims

1. 1. Use of one or more senescence inhibitors to increase survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

2. Use of one or more senescence inhibitors to increase the efficiency of gene editing of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells.

3. 1. One or more senescence inhibitors for use in hematopoietic cell gene therapy, hematopoietic stem cell gene therapy, hematopoietic progenitor cell gene therapy and / or T cell gene therapy.

4. 1. One or more senescence inhibitors for use in increasing survival and / or engraftment of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells in gene therapy.

5. The one or more aging inhibitors for the use of claim 1 or the use of claim 3 or claim 4, wherein the gene therapy comprises gene editing.

6. 5. The one or more aging inhibitors for use according to claim 1 or claim 2, or for use according to claim 3 or claim 4, wherein the one or more aging inhibitors comprise or consist of a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor.

7. a) the MAPK inhibitor is a JNK inhibitor, a p38 inhibitor or an ERK inhibitor; and / or b) the MAPK inhibitor is FR180204, SP600125, SB203580 or a derivative thereof; The use according to claim 6 comprises one or more anti-aging agents.

8. a) the IL-1 inhibitor is an anti-IL-1α antibody, an anti-IL-1β antibody, an IL-1 antagonist, an IL-1 receptor antagonist, an IL-1α converting enzyme inhibitor, an IL-1β converting enzyme inhibitor, or a soluble decoy IL-1 receptor; and / or b) the IL-1 inhibitor is IL-1Ra, anakinra, canakinumab, rilonacept, gevokizumab or a variant thereof; The use according to claim 6 or one or more anti-aging agents.

9. a) the NF-κB inhibitor is an IL-1 inhibitor, an IL-1 receptor inhibitor, a TLR4 inhibitor, a TAK1 inhibitor, an Akt inhibitor, an IKK inhibitor, an IκB phosphorylation inhibitor, an IκB degradation inhibitor, a proteasome inhibitor, an IκBα upregulation inhibitor, an NF-κB nuclear translocation inhibitor, an NF-κB expression inhibitor, an NF-κB DNA binding inhibitor, or an NF-κB transactivation inhibitor; and / or b) the NF-κB inhibitor is SC514 or a derivative thereof; IL-1Ra, anakinra, canakinumab, rilonacept, gevokizumab or a variant thereof; or siRNA, shRNA, miRNA or antisense DNA / RNA; The use according to claim 6 or one or more anti-aging agents.

10. 5. The use of claim 1 or claim 2, or one or more aging inhibitors for the use of claim 3 or claim 4, wherein the use further comprises the use of an agent that promotes homology-dependent DNA repair, optionally wherein the agent is a p53 activation inhibitor, and optionally wherein the p53 activation inhibitor is GSE56 or a mutant thereof.

11. 5. The one or more senescence inhibitors for the use according to claim 1 or claim 2, or the use according to claim 3 or claim 4, wherein the inhibition of senescence in the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells is transient, and optionally the MAPK inhibition, IL-1 inhibition and / or NF-κB inhibition in the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells is transient.

12. a) the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are exposed to the IL-1 inhibitor and / or NF-κB inhibitor before, simultaneously with and / or after a gene editing mechanism is introduced into the cells, optionally simultaneously with the introduction of the gene editing mechanism into the cells; and / or b) the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells are exposed to a MAPK inhibitor before the gene editing machinery is introduced into the cells; The use according to claim 6 or one or more anti-aging agents.

13. 5. The one or more senescence inhibitors for use according to claim 1 or claim 2, or for use according to claim 3 or claim 4, wherein the one or more senescence inhibitors are added to hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells at a concentration of about 0.5 to 200 μM or about 0.1 to 200 ng / μL.

14. 3. The use of claim 2, wherein the gene editing target is selected from the group consisting of FANC-A, CD40L, RAG-1, IL-2RG, CYBA, CYBB, NCF1, NCF2, and NCF4.

15. The use of claim 5 or one or more aging inhibitors, wherein the gene editing target is selected from the group consisting of FANC-A, CD40L, RAG-1, IL-2RG, CYBA, CYBB, NCF1, NCF2, and NCF4.

16. 1. A method for gene editing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells, comprising: (a) contacting said population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more senescence inhibitors; (b) introducing gene editing machinery into said population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells using one or more vectors; and (c) editing the genome of the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells, and / or T cells. A method comprising:

17. 1. A method for transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells using a viral vector, comprising: (a) contacting the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more senescence inhibitors; and (b) transducing said population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with one or more viral vectors. A method comprising:

18. 18. The method of claim 16 or claim 17, wherein the one or more senescence inhibitors comprise or consist of a MAPK inhibitor, an IL-1 inhibitor and / or an NF-κB inhibitor.

19. a) the population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells is contacted with a MAPK inhibitor prior to or simultaneously with the step of introducing gene editing machinery into or transducing the cells, optionally prior to the step of introducing gene editing machinery into or transducing the cells; and / or b) contacting the population of hematopoietic cells, hematopoietic stem cells and / or hematopoietic progenitor cells with an IL-1 inhibitor and / or an NF-κB inhibitor before, simultaneously with, or after introducing a gene editing mechanism into or transducing the cells; 18. The method of claim 16 or claim 17.

20. 18. The method of claim 16 or claim 17, further comprising contacting the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells with an agent that promotes homology-dependent DNA repair, optionally wherein the agent is a p53 activation inhibitor.

21. 18. The method of claim 16 or 17, wherein one or more senescence inhibitors are added to the hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells at a concentration of about 0.5 to 200 μM or about 0.1 to 200 ng / μL.

22. 18. The method of claim 16 or claim 17, wherein the population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells is obtained from mobilized peripheral blood, bone marrow or umbilical cord blood.

23. 18. The method of claim 16 or claim 17, comprising the further step of enriching said population for hematopoietic stem and / or progenitor cells and / or T cells.

24. 17. The method of claim 16, wherein the gene editing target is selected from the group consisting of FANC-A, CD40L, RAG-1, IL-2RG, CYBA, CYBB, NCF1, NCF2, and NCF4.

25. (a) gene editing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells according to the method of claim 16; and (b) administering the gene-edited hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells to a subject. A method of gene therapy comprising:

26. (a) transducing a population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells according to the method of claim 17; and (b) administering the population of transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells to a subject. A method of gene therapy comprising:

27. 18. A population of gene-edited and / or transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells prepared according to the method of claim 16 or claim 17.

28. 28. A pharmaceutical composition comprising the gene-edited and / or transduced population of hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells of claim 27.

29. 28. A population of gene edited and / or transduced hematopoietic cells, hematopoietic stem cells, hematopoietic progenitor cells and / or T cells according to claim 27 for use in therapy.

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