Methods and compositions for hematopoietic stem cell transplantation

Abstract

Provided are methods of and compositions useful for enhancing engraftment of donor hematopoietic stem cells (dHSC) in a recipient in utero subject. Such methods and compositions include one or more toxin-conjugated antibody that target and / or selectively bind to a hematopoietic stem cell, a lymphocyte, and / or a myeloid cell that is endogenous to the recipient in utero subject to enhance engraftment of dHSC in the recipient in utero subject.

Description

Attorney Docket No. 9865.247. WOMETHODS AND COMPOSITIONS FOR HEMATOPOIETIC STEM CELL TRANSPLANTATIONSTATEMENT OF PRIORITY

[0001] This application claims the benefit, under 35 U. S. C. 119(e), of U. S. Provisional Application No. 63 / 788,178 filed on April 14, 2025, the entire contents of which are incorporated by reference herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant Number R01HL 165247 awarded by the National Institutes of Health. The U. S. government has certain rights in the invention.STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0003] A Sequence Listing in XML format, entitled 9865-247WO _ST26.xml, 1,861 bytes in size, generated on April 13, 2026, and filed herewith, is hereby incorporated by reference into the specification for its disclosures.FIELD OF THE INVENTION

[0004] This invention relates to hematopoietic stem cell transplantation in a recipient in utero subject.BACKGROUND OF THE INVENTION

[0005] Clinical success using in utero transplantation (lUTx) of hematopoietic stem cells (HSCs) has been limited to patients with X-linked severe combined immunodeficiency (X-SCID). In X-SCID, the fetal recipient lacks functional lymphoid lineage cells, thereby conferring a selective advantage to donor HSCs, which can populate the deficient lineages and achieve durable engraftment. In contrast, many other disease targets proposed for lUTx — such as the hemoglobinopathies, including thalassemias and sickle cell disease (SCD) — do not provide such a selective advantage to the donor cells. As a result, donor HSCs must compete with an intact endogenous hematopoietic system, which presents a significant barrier to effective engraftment.Attorney Docket No. 9865.247. WO

[0006] Successful application of lUTx to a broader range of diseases requires some form of pre-transplantation conditioning to partially deplete the endogenous fetal HSC population, thereby creating hematopoietic niche space to accommodate donor-derived cells. In addition, the fetal immune system may be more immunologically competent than previously appreciated and may actively reject transplanted donor HSCs.

[0007] Conventional conditioning regimens used to facilitate donor cell engraftment in pediatric and adult patients employ ionizing radiation and / or cytotoxic chemotherapy, which are broadly genotoxic and non-selective. These agents target proliferative cells across multiple tissue types, including the gastrointestinal (GI) tract and other rapidly dividing somatic compartments. In addition to their systemic toxicity, these regimens confer a significant longterm risk of secondary malignancies due to DNA damage sustained by surviving cells. Consequently, such approaches are considered unacceptable for use in fetal subjects.

[0008] Efforts to develop more targeted conditioning approaches have included the use of antibody-drug conjugates (ADCs), which offer the potential to selectively deplete hematopoietic cells by coupling a cytotoxic payload to an antibody specific for a surface marker expressed on the target cell type. However, ADC-based approaches pose a risk of off-target toxicity, particularly in a fetal subject in which developmental processes are highly sensitive to cellular depletion in non-hematopoietic tissues.

[0009] Thus, there remains an unmet need for the development of safe, non-genotoxic conditioning regimens that enable robust and specific donor HSC engraftment following lUTx.SUMMARY OF THE INVENTION

[0010] In a first aspect, provided is a method of enhancing engraftment of donor hematopoietic stem cells (dHSC) in a recipient in utero subject, comprising: (a) depleting hematopoietic stem cells of the recipient in utero subject (rHSC) by administering to the recipient in utero subject a toxin-conjugated antibody targeting the rHSC; (b) depleting lymphocytes (e.g., NK, T and / or B cells) of the recipient in utero subject by administering a toxin-conjugated antibody targeting the lymphocytes; or (c) performing both (a) and (b).

[0011] In another aspect, provided is a method of enhancing engraftment of donor hematopoietic stem cells (dHSC) in a recipient in utero subject, comprising: (a) preparing a toxin-conjugated antibody targeting hematopoietic stem cells of the recipient in utero subject (rHSC); (b) preparing a toxin-conjugated antibody targeting lymphocytes (e.g., NK, T and / or B cells) of the recipient in utero subject; and (c) administering to the in utero subject the toxin-Attorney Docket No. 9865.247. WOconjugated antibody targeting the rHSC and / or the toxin-conjugated antibody targeting the lymphocytes.

[0012] In some embodiments, the toxin-conjugated antibody of (a) and / or (b) comprises an antibody covalently or non-covalently linked to a nanoparticle (e.g., a lipid nanoparticle). In some embodiments, the nanoparticle is a lipid nanoparticle comprising mRNA encoding the toxin.

[0013] In some embodiments, the toxin of (a) and / or (b) is saporin, Pseudomonas exotoxin A (PE), Diphtheria toxin (DT), ricin A chain, gelonin, bouganin, abrin A chain, colicin E3, alpha-sarcin, and / or BLF1 (Burkholderia lethal factor 1). In some embodiments, the toxin of (a) and / or (b) is alpha-Amanitin, MMAE (monomethyl auristatin E), or the catalytic domain of Pseudomonas exotoxin A (PE).

[0014] In some embodiments, the toxin-conjugated antibody of (a) targets (e.g., selectively binds to) GPI-80 (glycosylphosphatidylinositol-anchored protein 80, also known as VNN2).

[0015] In some embodiments, the toxin-conjugated antibody of (b) targets (e.g., selectively binds to) CD56, CD335 and / or IL2RG.

[0016] In some embodiments, the toxin-conjugated antibody of (a) and / or (b) also targets and depletes myeloid cells of the recipient in utero subject.

[0017] In some embodiments, the method further comprises engrafting of the donor hematopoietic stem cells (dHSC) in the recipient in utero subject.

[0018] In some embodiments, the method further comprises, prior to the engrafting of the dHSC in the recipient in utero subject, enhancing dHSC homing to a desired hematopoietic niche of the recipient in utero subject, thereby further enhancing engraftment of dHSC. In some embodiments, the enhancing dHSC homing comprises, prior to the engrafting of the dHSC in the recipient in utero subject, reducing expression of an enzyme (e.g., a cell-surface protease, such as peptidyl-peptidase) of the dHSC to increase responsiveness of the dHSC to chemokine signaling directing the dHSC to the desired hematopoietic niche. In some embodiments, the desired hematopoietic niche is bone marrow and / or liver of the recipient in utero recipient.

[0019] In some embodiments, the method further comprises, prior to the engrafting of the dHSC in the recipient in utero subject, enriching for dHSCs that express CD 166.

[0020] In another aspect, provided is a composition useful for enhancing engraftment of donor hematopoietic stem cell (dHSC) in a recipient in utero subject, comprising: a first toxin-conjugated antibody that targets an extracellular domain of a hematopoietic stem cell that is endogenous to the recipient in utero subject (rHSC); and / or, a second toxin-conjugated antibody that targets an extracellular domain of a lymphocyte that is endogenous to theAttorney Docket No. 9865.247. WOrecipient in utero subject, wherein the composition comprises a pharmaceutically acceptable carrier.

[0021] In some embodiments, the first toxin-conjugated antibody and / or the second toxin-conjugated antibody comprises an antibody covalently or non-covalently linked to a nanoparticle (e.g., a lipid nanoparticle). In some embodiments, the nanoparticle is a lipid nanoparticle comprising mRNA encoding the toxin.

[0022] In some embodiments, the toxin of the first toxin-conjugated antibody and / or the second toxin-conjugated antibody is saporin, Pseudomonas exotoxin A (PE), Diphtheria toxin (DT), ricin A chain, gelonin, bouganin, abrin A chain, colicin E3, alpha-sarcin, alpha-Amanitin, MMAE (monomethyl auristatin E), and / or BLF1 (Burkholderia lethal factor 1).

[0023] In some embodiments, the toxin of the first toxin-conjugated antibody and / or the second toxin-conjugated antibody is alpha-Amanitin, MMAE (monomethyl auristatin E), or the catalytic domain of Pseudomonas exotoxin A (PE).

[0024] In some embodiments, the first toxin-conjugated antibody targets (e.g., selectively binds to) GPI-80 (glycosylphosphatidylinositol-anchored protein 80, also known as VNN2).

[0025] In some embodiments, the second toxin-conjugated antibody targets (e.g., selectively binds to) CD56, CD335, and / or IL2RG.

[0026] In some embodiments, the first and / or second toxin-conjugated antibody also targets an extracellular domain of a myeloid cell that is endogenous to the recipient in utero subject.

[0027] Further provided is the use of a composition as taught herein in a method of enhancing engraftment of donor hematopoietic stem cells (dHSC) in a recipient in utero subject, or for the manufacture of a medicament for enhancing engraftment of donor hematopoietic stem cells (dHSC) in a recipient in utero subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 illustrates an example method for producing and purifying a recombinant version of the type I ribosome-inactivating protein toxin saporin and subsequent bioconjugation to streptavidin, enabling production of a cell-specific immunotoxin by simple linkage to a biotinylated antibody to the desired target protein.

[0029] FIG. 2 presents example microscopy images reflecting reduced expression of c-Kit (CD117) in early hematopoietic cells seeding the fetal human bone marrow (CD45+). CD117+ co-expresses Stro-1, indicating they are of desired hematopoietic niche origin.Attorney Docket No. 9865.247. WO

[0030] FIG.3 shows data supporting that co-transplantation of mesenchymal stem cells with HSC enhances the level of donor HSC following lUTx. Such enhancement occurs irrespective of whether the MSC are transplanted 3 days prior to the HSC or simultaneously.

[0031] FIG. 4 shows data reflecting transplantation of MSC alone markedly reduced the levels of CD4+, CD8+, and CD4+CD8+ T cells in the recipient when compared to non-IUTx age-matched controls. Transplantation of mesenchymal stem cells in utero leads to reduction in fetal T cells, thereby minimizing a barrier to HSC engraftment.

[0032] FIG. 5 illustrates an overview of Experiment 1, as described herein. For each condition, n=16 fetuses at 55-57 gestational days; A indicates n=8 which were euthanized 60 days post-IUTx; and B indicates n=8 which were followed for 6 months post-birth.

[0033] FIG. 6 shows how fetal HSC with primitive clonogenic potential reside in CD34-CD166+ cell populations. During fetal ontogeny, the majority of the primitive HSC leaving the fetal liver to seed the nascent bone marrow do not express CD34. Instead, most of these HSC express the surface marker CD166 / ALCAM.

[0034] FIGs. 7A-C illustrate a correlation between levels of donor-derived CD146+ cells expressing SDF-1 that engraft within vasculature and levels of donor derived GRF+ hematopoietic cells present in the perivasculature following lUTx.

[0035] FIG. 8 illustrates an overview of Experiment 2, as described herein. Competitive lUTx comparing engraftment potential of CD34+ vs. CD 166+ HSC in utero (top) and the impact of shRNA-mediated knockdown of CD26 on donor HSC (bottom).

[0036] FIGs. 9A-C show data supporting that GPI-80- (FIG. 9A), CD335- (FIG. 9B), and IL2RG-based (FIG.9C) antibody-drug conjugates (ADCs) are efficiently internalized in sheep peripheral blood and bone marrow mononuclear cells (PBMCs and BMNCs) in vitro.

[0037] FIGs. 10A-C show data supporting that GPI-80- (FIG. 10A), CD335- (FIG. 10B), and IL2RG-based (FIG. 10C) antibody-drug conjugates (ADCs) mediate the cytotoxic killing of sheep PBMCs and BMNCs in vitro.

[0038] FIGs. 11A-F show levels of GPI-80, c-Kit, IL2RG, and CD335 in fetal sheep non-hematopoietic organs.

[0039] FIG. 12 shows immunohistochemistry staining images illustrating the expression of GPI-80, c-kit IL2RG, and CD335 in fetal sheep non-hematopoietic organs.

[0040] FIGs. 13A-D show levels of GPI-80, c-Kit, IL2RG, and CD335 in fetal sheep hematopoietic tissues.

[0041] FIG. 14 shows immunohistochemistry staining images illustrating the expression of GPI-80, c-kit IL2RG, and CD335 in fetal sheep hematopoietic tissues.Attorney Docket No. 9865.247. WO

[0042] FIGs. 15A-D show data reflecting that ADCs facilitate the engraftment of human donor bone marrow (13A) cells; peripheral blood (13B) cells; human CD33+ cells (13C); and human CD34+ cells (13D).

[0043] FIGs. 16A-D show data supporting that conditioning with either GPI-80, IL2RG, or CD335 does not cause hepatic or hematological alterations.DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0045] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0047] The term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.Attorney Docket No. 9865.247. WO

[0048] As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0049] Also, as used herein, “one or more” or “at least one” means one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.

[0050] As used herein, the term “subject” can refer to any animal, including, but not limited to, humans and non-human animals (e.g., rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, etc.). The terms “patient” and “subject” may be used interchangeably herein in reference to a human subject.

[0051] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0052] As used herein, the transitional phrase “consisting essentially of’ is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0053] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelvefold, or even fifteen-fold.

[0054] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0055] A “therapeutically effective” or “treatment effective” amount as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a “therapeutically effective” or “treatment effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject (e.g., in the case of Sickle Cell Disease (SCD), or any other genetic disorder that may be treated in utero). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0056] By the term “treat,” “treating,” or “treatment of’ (or grammatically equivalent terms) is meant to reduce or to at least partially improve or ameliorate the severity of the subject’sAttorney Docket No. 9865.247. WOcondition and / or to alleviate, mitigate or decrease in at least one clinical symptom and / or to delay the progression of the condition.

[0057] As used herein, the term “prevent,” “prevents,” or “prevention” (and grammatical equivalents thereof) means to delay or inhibit the onset of a disease. The terms are not meant to require complete abolition of disease and encompass any type of prophylactic treatment to reduce the incidence of the condition or delay the onset of the condition.

[0058] Any nucleotide sequences are presented herein by single strand only, in the 5’ to 3’ direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C. F. R. §1.822 and established usage.

[0059] Except as otherwise indicated, standard methods known to those skilled in the art may be used for production of recombinant and synthetic polypeptides, antibodies or antigenbinding fragments thereof, manipulation of nucleic acid sequences, production of transformed cells, the construction of rAAV constructs, modified capsid proteins, packaging vectors expressing the AAV rep and / or cap sequences, and transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY ANUAL 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Assoc., Inc., and John Wiley & Sons, Inc., NY, 1987).

[0060] As used herein, the terms “protein” and “polypeptide” are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.

[0061] The term “consists essentially of,” and any grammatical variants thereof, as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5’ and / or 3’ or N-terminal and / or C-terminal ends of the recited sequence or between the two ends (e.g., between domains) such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added together. The term “materially altered,” as applied to polynucleotides of the invention, refers to an increase or decrease in ability to express the encoded polypeptide of at least about 50% or more as compared to the expression level of a polynucleotide consisting of the recited sequence. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activity of atAttorney Docket No. 9865.247. WOleast about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0062] The term “fragment,” as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and / or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention.

[0063] As used herein, an “isolated” polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.

[0064] As used herein, the term “modified,” as applied to a polypeptide sequence, refers to a sequence that differs from a wildtype sequence due to one or more deletions, additions, substitutions, or any combination thereof.

[0065] “Pharmaceutically acceptable,” as used herein, means a material that is not biologically or otherwise undesirable, / .<., the material can be administered to an individual along with the compositions of this invention, without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art (see, e.g., Remington's Pharmaceutical Science,- 21sted. 2005). In some embodiments, the pharmaceutically acceptable carrier is a physiological buffer such that it maintains the composition in a physiological pH range (e.g., a pH range between about 7.00 and about 7.50, (e.g., about 7.00, 7.05, 7.10, 7.15, 7.20, 7.25, 7.30, 7.35, 7.40, 7.45, or 7.50)). Exemplary physiological buffers for pharmaceutically acceptable carriers for the compositions of this invention include, but are not limited to, phosphate buffered saline (PBS), HEPES, sterile pyrogen-free water, and other sterile pyrogen-free physiological saline solutions.Attorney Docket No. 9865.247. WO

[0066] The term “administering” or “administration” of a composition of the present invention to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function (e.g., for conditioning hematopoietic stem cells (HSC), such as HSCs for transplantation in a recipient in utero subject). In some embodiments, the administration comprises intravenous administration, mucosal administration, intradermal administration, intramuscular administration, vaginal administration, rectal administration, subcutaneous administration, transdermal administration, oral administration, sublingual administration, intranasal administration, and / or intratumoral administration.

[0067] In some embodiments, HSCs may be administered to a recipient in utero subject in a therapeutically effective quantity, volume, or concentration. In some embodiments, a therapeutically effective concentration of HSC may be about 106cells / kg, 107cells / kg, 108cells / kg, 109cells / kg, IO10cells / kg, 1011cells / kg, 1012cells / kg, wherein kg is in reference to the mass of a recipient in utero subject. In some embodiments, a therapeutically effective concentration of HSC may be from about from about 106cells / kg to about 108cells / kg, from about 107cells / kg to about 109cells / kg, from about 108cells / kg to about IO10cells / kg, from about 109cells / kg to about 1011cells / kg, from about IO10cells / kg to about 1012cells / kg, wherein kg is in reference to the mass of a recipient in utero subject. In some embodiments, the concentration of dHSC may be suspended in a liquid volume, e.g., about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, or any range of volumes therebetween. In some embodiments, administration of HSC to the recipient in utero subject may be at any location therein by any suitable method as would be appreciated by one of ordinary skill in the relevant art.

[0068] A “sample,” “biological sample,” and / or “ex vivo sample” of the present invention may be any biological material, such as a biological fluid, an extract from a cell, an extracellular matrix isolated from a tissue, a cell (in solution or bound to a solid support), a tissue, a tissue homogenate, and the like as are well known in the art.

[0069] As used herein, “zw utero" refers to conditions, processes, or interventions occurring within the uterus of a pregnant female, specifically during the gestational period prior to birth. The term may refer to any stage of fetal development within the maternal uterine environment, including embryonic and fetal stages, and may pertain to diagnostic, therapeutic, or monitoring procedures applied directly or indirectly to the fetus while it remains in the uterus.

[0070] As used herein “antibody” refers to a glycoprotein molecule produced by B cells that specifically binds to an antigen. Antibodies may be composed of two identical heavy (H) chains and two identical light (L) chains connected by disulfide bonds to form a Y-shaped structure.Attorney Docket No. 9865.247. WOEach chain may include a variable region (V) that determines antigen specificity, and a constant region (C) that defines the antibody isotype (e.g., IgG, IgA, IgM, IgD, IgE). The antigenbinding region may be formed by the variable domains of the heavy and light chains (VH and VL), which together constitute the Fab (fragment antigen-binding) portion of the molecule, while the Fc (fragment crystallizable) region may mediate effector functions by interacting with Fc receptors (FcRs) and complement proteins. Antibodies may include whole antibodies, as well as antibody fragments and derivatives that retain specific antigen-binding capability, including but not limited to Fab fragments, F(ab’)2 fragments, single-chain variable fragments (scFv), diabodies, triabodies, nanobodies, and domain antibodies (dAbs). Antibodies may include engineered antibodies, such as humanized antibodies, chimeric antibodies, bispecific antibodies, multivalent or multispecific antibodies, antibody-drug conjugates (ADCs), antibody-lipid nanoparticles (Ab-LNPs), and antibodies modified to alter glycosylation, effector function, or pharmacokinetics. Antibodies may include antibodies to which a moiety (e.g., a pharmaceutical composition) is conjugated, including toxin-conjugated antibodies, which may include antibodies covalently or non-covalently linked to an effector moiety, such as a cytotoxic agent (e.g., toxin), radionuclide, enzyme, imaging agent, therapeutic payload, or nanoparticle (e.g., a lipid nanoparticle, for example a lipid nanoparticle comprising mRNA encoding a cytotoxic agent (e.g., toxin), enzyme, imaging agent, or therapeutic payload)), wherein the antibody serves to target delivery of the conjugated moiety to a specific antigenexpressing cell or tissue.

[0071] As used herein, “non-genotoxic” refers to a compound, agent, or pharmaceutical composition that does not cause substantial damage to DNA or induce genetic mutations, chromosomal aberrations, or other forms of genetic instability under intended conditions of use. A non-genotoxic composition may be a composition that does not initiate or promote genotoxic mechanisms, such as DNA strand breaks, base modifications, crosslinking, or interference with normal DNA replication or repair processes. In the context of a pharmaceutically acceptable composition, “non-genotoxic” may indicate that the formulation is suitable for administration to a subject without posing a significant risk of mutagenesis or carcinogenesis due to direct interaction with the genome.

[0072] As used herein, “hematopoietic stem cells” (HSCs or HSC) refers to multipotent stem cells capable of self-renewal and differentiation into all lineages of mature blood cells, including erythrocytes, leukocytes (both myeloid and lymphoid lineages), and platelets. HSCs may be characterized by their ability to reconstitute hematopoiesis in a myeloablated host and are identifiable by surface markers, such as CD34+ in humans, although marker expressionAttorney Docket No. 9865.247. WOmay vary depending on developmental stage, source, and species. As used herein, “donor hematopoietic stem cells” or “dHSCs” refers to HSCs derived from an ex utero donor source, including but not limited to peripheral blood, bone marrow, or mobilized peripheral blood of a postnatal (adult or pediatric) donor. dHSCs may be autologous, allogeneic, or xenogeneic relative to the recipient, and may be naive, genetically modified, or otherwise manipulated ex vivo prior to administration. As used herein, “recipient in utero hematopoietic stem cells” or “rHSCs” refers to an endogenous hematopoietic stem cells present within a developing fetus (e.g., the recipient in utero subject). These cells may reside in various anatomical compartments depending on gestational age, including the yolk sac, aorta-gonad-mesonephros (AGM) region, fetal liver, and eventually the developing bone marrow. rHSCs may be capable of responding to signals from exogenous HSCs and may be involved in niche competition, immune tolerance, or hematopoietic reconstitution following transplantation.

[0073] As used herein, “conditioning” refers to one or more interventions administered to a recipient in utero subject prior to, or concurrently with, the introduction of donor hematopoietic stem cells (dHSCs), to facilitate engraftment, promote niche availability, modulate recipient immune response, enhance the homing and expansion of donor cells, and the like. Conditioning may involve non-genotoxic or genotoxic approaches, including but not limited to, depletion or suppression of endogenous recipient hematopoietic stem cells (rHSCs), modulation of the fetal immune or stromal microenvironment, transient disruption of the hematopoietic niche, or alteration of cytokine and chemokine signaling pathways (e.g., SDF-1 / CXCR4 axis). Examples of conditioning agents or techniques can include antibodies or other biologies that are known in the relevant art for targeting cell surface markers (e.g., anti-c-Kit or anti-CD117), small molecules, enzymes, cytokines, or other agents that selectively deplete or inactivate rHSCs or immune effector cells. In some embodiments, conditioning may be non-myeloablative or minimally toxic, particularly to preserve fetal viability and minimize off-target effects.

[0074] As used herein, “sickle cell disease” or “SCD” refers to a group of inherited hemoglobinopathies caused by mutations in the β-globin gene (HBB), most commonly a single nucleotide substitution (Glu6Val) resulting in the production of hemoglobin S (HbS). Under deoxygenated conditions, HbS polymerizes, causing red blood cells to adopt a characteristic rigid, sickle-like shape. These deformed erythrocytes exhibit reduced deformability, increased hemolysis, and a propensity to adhere to endothelium, leading to vaso-occlusion, chronic hemolytic anemia, and systemic ischemia-reperfusion injury. SCD is a monogenic, autosomal recessive disorder and encompasses several genotypes, including HbSS (homozygous for HbS), as well as compound heterozygous forms such as HbSC, HbS / β°-thalassemia, andAttorney Docket No. 9865.247. WOHbS / β+-thalassemia, all of which present with variable clinical severity. SCD may be characterized by acute and chronic complications, including pain crises, stroke, acute chest syndrome, splenic sequestration, and end-organ damage. In the context of therapeutic intervention, treatment strategies using, e.g., dHSCs, may aim to reduce sickling, increase fetal hemoglobin (HbF), correct or replace the defective hematopoietic stem cell population, or modulate downstream pathophysiological pathways.

[0075] It will be appreciated by one having ordinary skill in the relevant art, that the present disclosure may relate to any hematological disorder that may be treated in a recipient in utero subject using dHSCs. Such disorders include, but are not limited to, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, chronic granulomatous disease, X-linked lymphoproliferative disease, leukocyte adhesion deficiency, Fanconi anemia, Diamond-Blackfan anemia, beta-thalassemia, alpha-thalassemia major, sickle cell disease, Gaucher disease, Hurler syndrome (MPS I), Krabbe disease, Tay-Sachs disease, adenosine deaminase deficiency (ADA-SCID), osteopetrosis, Nijmegen breakage syndrome, and the like. See, e.g., Muench (2005) Bone Marrow Transplantation, 35, 537-547.

[0076] As used herein, “engraftment” refers to the migration, homing, and / or long-term functional integration of transplanted hematopoietic stem cells (HSCs) into a hematopoietic niche (e.g., bone marrow and / or liver) of a recipient (e.g., recipient in utero subject), wherein the donor HSCs persist and give rise to multilineage hematopoiesis. Engraftment may involve several stages or steps, including homing of HSCs to the appropriate microenvironment, retention within the niche, proliferation, and differentiation into one or more mature blood cell lineages (e.g., myeloid, erythroid, lymphoid). In the context of an in utero recipient, engraftment may include a durable presence of donor-derived hematopoietic cells in fetal or neonatal peripheral blood, bone marrow, liver, or other hematopoietic tissues, as determined, e.g., by chimerism analysis (e.g., flow cytometry, PCR-based genotyping, or lineage-specific markers). Engraftment may be transient, partial, or durable and multilineage, depending on the efficiency of niche occupancy, immune compatibility, and conditioning regimen used. For the purposes of this disclosure, durable engraftment typically refers to the persistence of donor-derived cells over an extended period (e.g., weeks to months or longer) with evidence of sustained hematopoietic function contributed by the transplanted HSCs. In some embodiment, engraftment of dHSCs in a recipient in utero subject may be about 5%, 10%, 15%, 20%, or 25% in the recipient in utero subject. In some embodiments, engraftment of dHSCs in a recipient in utero subject may be about from about 5% to about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25% in the recipient in uteroAttorney Docket No. 9865.247. WOsubject. For example, percentage engraftment can be used to express “successful engraftment,” wherein, e.g., engraftment of 20% can be considered to be successfully engrafting dHSCs in a recipient in utero subject.

[0077] As used herein, “depleting,” and grammatical variants thereof, refers to the process of reducing the number, viability, and / or functional capacity of a cell or population of cells (e.g. rHSCs) in a recipient in utero subject niche, such as the bone marrow and / or liver thereof. Depletion may be achieved by inducing cell death (e.g., apoptosis or necrosis), inhibiting proliferation or self-renewal, promoting differentiation to remove cells from the stem or progenitor pool, mobilizing cells out of their hematopoietic niche, or blocking essential survival or signaling pathways. In the context of preparing a recipient in utero subject for transplantation of dHSCs, depleting rHSCs may facilitate engraftment by creating physical space within the hematopoietic niche, reducing cellular competition, and / or mitigating immune-mediated barriers to donor cell persistence. Such depletion may be partial or complete and may be accomplished using biological agents (e.g., monoclonal antibodies targeting cell surface markers), small molecules, cytokines, or other pharmacologic or molecular interventions.

[0078] In some embodiments, a hematopoietic niche may be depleted of rHSCs such that the hematopoietic niche is about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% depleted of rHSCs. In some embodiments, the hematopoietic niche may be from about from about 100% to about 95%, from about 90% to about 85%, from about 80% to about 75%, from about 70% to about 65%, from about 60% to about 55%, from about 50% to about 45%, from about 40% to about 35%, from about 30% to about 25%, from about 20% to about 15%, or from about 10% to about 5% depleted of rHSCs. In some embodiments, depleting may be performed about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days prior to administering dHSCs to a recipient in utero donor subject. In some embodiments, depleting may be performed from about from about 5 days to about 7 days, from about 6 days to about 8 days, from about 7 days to about 9 days, from about 8 days to about 10 days, from about 9 days to about 11 days, from about 10 days to about 12 days, from about 11 days to about 13 days, from about 12 days to about 14 days, from about 13 days to about 15 days prior to administering dHSCs to a recipient in utero donor subject.

[0079] As used herein, the term “to target,” and grammatical variants thereof, refers to the ability of a molecule, composition, or delivery system (e.g., an antibody, ligand, nanoparticle, or engineered cell) to preferentially direct or deliver an effector function, agent, or payload toAttorney Docket No. 9865.247. WOa specific cell type, tissue, or molecular structure based on the presence of a distinguishable marker, receptor, or microenvironmental characteristic. Targeting may involve physical, biochemical, or affinity-based mechanisms and does not necessarily require direct molecular binding to the target structure but may include it. In contrast, “to selectively bind,” and grammatical variants thereof, as used herein refers to the preferential and specific physical interaction (e.g., non-covalent binding) of one molecule (such as an antibody or ligand) with a particular epitope, antigen, or molecular locus relative to other non-target molecules. Selective binding may include a higher binding affinity for the intended locus compared to unrelated or structurally similar molecules, thereby minimizing off-target interactions under physiological conditions. Thus, while selective binding may describe a molecular interaction based on affinity and specificity, targeting may describe a broader concept that includes the direction or localization of an agent to a desired site, which may or may not rely on selective binding.

[0080] Disclosed herein is a method of enhancing engraftment of donor hematopoietic stem cells (dHSC) in a recipient in utero subject, the method may include the steps of (a) depleting hematopoietic stem cells of the recipient in utero subject (rHSC) by administering to the recipient in utero subject a toxin-conjugated antibody targeting the rHSC; (b) depleting lymphocytes (e.g., NK, T and / or B cells) of the recipient in utero subject by administering a toxin-conjugated antibody targeting the lymphocytes; or (c) performing both (a) and (b).

[0081] Also disclosed herein is a method of enhancing engraftment of donor hematopoietic stem cells (dHSC) in a recipient in utero subject, the method may include the steps of (a) preparing a toxin-conjugated antibody targeting hematopoietic stem cells of the recipient in utero subject (rHSC); (b) preparing a toxin-conjugated antibody targeting lymphocytes (e.g., NK, T and / or B cells) of the recipient in utero subject; and (c) administering to the in utero subject the toxin-conjugated antibody targeting the rHSC and / or the toxin-conjugated antibody targeting the lymphocytes.

[0082] In some embodiments, a toxin-conjugated antibody used in a method of the present invention includes an antibody covalently or non-covalently linked to and / or otherwise associated with a toxin. In some embodiments, a toxin-conjugated antibody used in a method of the present invention includes an antibody covalently or non-covalently linked to a nanoparticle that may comprise the toxin. In some embodiments, the nanoparticle is a lipid nanoparticle (e.g., comprising lipid 10 (an ionizable amino lipid)). In some embodiments, the nanoparticle (e.g., lipid nanoparticle) comprises (e.g., encapsulates) mRNA encoding the toxin, whereby delivery of the mRNA into target cells has intracellular toxicity upon mRNA translation.Attorney Docket No. 9865.247. WO

[0083] In some embodiments, the nanoparticle comprises a CD47 peptide. In some embodiments, the CD47 peptide comprises the sequence of GNYTCEVTELSREGKTVIELK (SEQ ID NO:1)

[0084] In some embodiments, the nanoparticle comprises glutamate-cholesterol (e.g., in place of cholesterol).

[0085] In some embodiments, administering (e.g. in human recipient in utero subjects) may be performed at 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks of gestation. In some embodiments, administering (e.g. in human recipient in utero subjects) may be performed at from about 16 weeks to about 18 weeks, from about 17 weeks to about 19 weeks, from about 18 weeks to about 20 weeks, from about 19 weeks to about 21 weeks, from about 20 weeks to about 22 weeks, from about 21 weeks to about 23 weeks, from about 22 weeks to about 24 weeks, from about 23 weeks to about 25 weeks, from about 24 weeks to about 26 weeks of gestation.

[0086] In some embodiments, a toxin-conjugated antibody may be administered to the recipient in utero subject about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days prior to administering dHSCs to a recipient in utero donor subject. In some embodiments, a toxin-conjugated antibody may be administered to the recipient in utero subject from about 5 days to about 6 days, from about 6 days to about 7 days, from about 7 days to about 8 days, from about 8 days to about 9 days, from about 9 days to about 10 days, from about 10 days to about 11 days, from about 11 days to about 12 days, from about 12 days to about 13 days, from about 13 days to about 14 days, from about 14 days to about 15 days prior to administering dHSCs to a recipient in utero donor subject. For example, a saporin-conjugated antibody can be administered about 5-10 days prior to dHSC transplantation in a recipient in utero subject.

[0087] In some embodiments, the lymphocytes may be one or more NK cell. For example, the method can include depleting one or more NK cell types, while having no effect on other lymphocyte cell types. In some embodiments, the lymphocytes may be one or more NK cell, T cell, and / or B cell. For example, the method can include depleting one or more types of lymphocytes, including, but not limited to, NK cells, T cells, B cells, and the like. In some embodiments, a toxin-conjugated antibody targeting lymphocytes may also target and deplete one or more myeloid cell types of a recipient in utero subject. In some embodiments, the myeloid cells may be one or more neutrophil, monocyte, macrophage, dendritic cell, eosinophil, basophil, mast cell, and the like. Thus, the method can include depleting one orAttorney Docket No. 9865.247. WOmore types of myeloid cells, including, but not limited to, neutrophils, monocytes, macrophages, dendritic cells, eosinophils, basophils, mast cells, and the like.

[0088] In some embodiments, a toxin used with a toxin-conjugated antibody targeting an rHSC may include a toxin that is non-cytotoxic until cleaved from an antibody to which it is linked. In some embodiments, the toxin may be non-cytogenic until said toxin is internalized within a cell lysosome or like organelles. In some embodiments, the toxin may be one or more of saporin, Pseudomonas exotoxin A (PE), Diphtheria toxin (DT), ricin A chain, gelonin, bouganin, abrin A chain, colicin E3, alpha-sarcin, BLF1 (Burkholderia lethal factor 1), or the like, for example, including, but not limited to, toxins disclosed by Griffin et al. (2022) J. of Hematology & Oncology, 16:65.

[0089] In some embodiments, a toxin-conjugated antibody targeting a rHSC may target a surface protein of an HSC. In some embodiments, a toxin-conjugated antibody targeting a rHSC may selectively bind to a surface protein of an HSC. In some embodiments, a toxin-conjugated antibody targeting a rHSC may target (or selectively bind to) a receptor tyrosine kinase (e.g., c-Kit (CD117)). In some embodiments, a toxin-conjugated antibody targeting a rHSC may target (or selectively bind to) a glycosylphosphatidylinositol (GPI)-anchored protein, e.g. a GPI-80 (VNN2). In some embodiments, a toxin-conjugated antibody targeting a rHSC may target (or selectively bind to) both a receptor tyrosine kinase (e.g., c-Kit) and a (GPI)-anchored protein (e.g., GPI-80). In some embodiments, a same toxin-conjugated antibody targeting a rHSC may target (or selectively bind to) a receptor tyrosine kinase (e.g., c-Kit) and a (GPI)-anchored protein (e.g., GPI-80). In some embodiments, a same toxin-conjugated antibody targeting a rHSC may target (or selectively bind to) both a receptor tyrosine kinase (e.g., c-Kit) and a GPI-anchored protein (e.g., GPI-80), wherein the first toxin-conjugated antibody targets exclusively to only one of the two targets. For example, a first toxin-conjugated antibody can target (or selectively bind to) c-Kit and GPI-80, but once c-Kit (or GPI-80) is targeted (or selectively bound) the GPI-80 (or c-Kit, respectively) can no longer be targeted (or selectively bound). In some embodiments, when a toxin-conjugated antibody targets (or selectively binds to) a GPI-anchored protein (e.g., GPI-80), the toxin-conjugated antibody may target both lymphocytes and myeloid cells.

[0090] In some embodiments, a toxin-conjugated antibody may target (or selectively bind to) a cell surface protein of a lymphocyte. In some embodiments, a toxin-conjugated antibody may target (or selectively bind to) a cell surface protein of a myeloid cell. In some embodiments, a toxin-conjugated antibody targeting lymphocytes and / or myeloid cells may target (or selectively bind to) CD56. In some embodiments, a toxin-conjugated antibody targetingAttorney Docket No. 9865.247. WOlymphocytes and / or myeloid cells may target (or selectively bind to) CD335. In some embodiments, a toxin-conjugated antibody targeting lymphocytes and / or myeloid cells may target (or selectively bind to) IL2RG. In some embodiments, a toxin-conjugated antibody targeting lymphocytes and / or myeloid cells may target (e.g., selectively bind to) CD56, CD335, and / or IL2RG.

[0091] In some embodiments, a method of enhancing engraftment of a dHSC in a recipient in utero subject may further comprise engrafting of the donor hematopoietic stem cells (dHSC) in the recipient in utero subject. In some embodiments, a method of enhancing engraftment of a dHSC in a recipient in utero subject may further comprise, prior to engrafting of the dHSC in the recipient in utero subject, enhancing dHSC homing to a desired hematopoietic niche. For example, enhancing dHSC homing can further enhance engraftment of dHSC by reducing expression of an enzyme (e.g., a cell-surface protease, such as peptidyl-peptidase) expressed on the cell surface of a dHSC to increase responsiveness thereof to chemokine signaling that can enhance directing the dHSC to the desired hematopoietic niche. In some embodiments, enhancing dHSC homing may comprise, prior to engrafting of the dHSC in the recipient in utero subject, reducing expression of an enzyme (e.g., a cell-surface protease, such as, but not limited to, peptidyl-peptidase) of the dHSC to increase responsiveness of the dHSC to chemokine signaling directing the dHSC to the desired hematopoietic niche. In some embodiments, a method of enhancing engraftment of a dHSC in a recipient in utero subject may include administering a vector (e.g. a viral vector, such as, but not limited to, a lentiviral vector) encoding shRNA to reduce (e.g., knockdown) CD26 expression in dHSCs to enhance SDF-1 -mediated homing to a desired hematopoietic niche. In some embodiments, a method of enhancing engraftment of a dHSC in a recipient in utero subject may include administering CD26-targeting shRNA in dHSCs to improve responsiveness to desired hematopoietic niche-derived hematopoietic growth factors. In some embodiments, a method of enhancing engraftment of a dHSC in a recipient in utero subject may include administering donor hematopoietic stem cells transduced with a lentivirus encoding CD26-specific shRNA to increase chemotactic migration and engraftment efficiency in a desired hematopoietic niche. For example, a lentiviral-driven shRNA can be used to knockdown expression of the peptidyl-peptidase CD26 on a dHSC to increase SDF-1 driven homing and responsiveness to niche-derived hematopoietic growth factors. In some embodiments, a desired hematopoietic niche can include bone marrow, peripheral blood, umbilical cord blood, fetal liver, spleen, aorta-gonad-mesonephros (AGM) region, placenta, and the like.Attorney Docket No. 9865.247. WO

[0092] In some embodiments, dHSCs may include dHSCs that express one or more cell surface receptor. In some embodiments, the dHSC may include dHSCs that express CD 116, CD130, CD117, CD135, CD123, CD25, CD127, CD116, CD119, CD105, CD184, CD90, and the like. In some embodiments, the dHSC may include dHSCs that express CD116. In some embodiments, dHSCs may include an enriched population of dHSCs that express a specific cell surface receptor (e.g., CD116). In some embodiments, dHSCs may comprise a population of CD166-expressing dHSCs. It will be understood by one having skill in the relevant art that, prior to engrafting of the dHSC in the recipient in utero subject, dHSCs may be enriched for or a subset of dHSCs expressing one or more cell surface receptor using conventional means in the relevant art, including, but not limited to, flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), immunomagnetic bead separation, microfluidic cell sorting, immunoaffinity chromatography, and the like.

[0093] Disclosed herein is a composition useful for enhancing engraftment of donor hematopoietic stem cells (dHSC) in a recipient in utero subject, the composition may comprise: a first toxin-conjugated antibody that targets an extracellular domain of a hematopoietic stem cell that is endogenous to the recipient in utero subject (rHSC); and / or, a second toxin-conjugated antibody that targets an extracellular domain of a lymphocyte that is endogenous to the recipient in utero subject, wherein the composition comprises a pharmaceutically acceptable carrier. In some embodiments, the second toxin-conjugated antibody also targets an extracellular domain of a myeloid cell that is endogenous to the recipient in utero subject.

[0094] In some embodiments, a toxin of a first toxin-conjugated antibody and / or a second toxin-conjugated antibody may comprise a toxin that is non-cytotoxic until cleaved from an antibody to which it is linked. In some embodiments, the toxin may be non-cytogenic until said toxin is internalized within a cell lysosome or like organelles. In some embodiments, the toxin may be one or more of saporin, Pseudomonas exotoxin A (PE), Diphtheria toxin (DT), ricin A chain, gelonin, bouganin, abrin A chain, colicin E3, alpha-sarcin, BLF1 (Burkholderia lethal factor 1), or the like, for example, including, but not limited to, toxins disclosed by Griffin et al. (2022) J. of Hematology & Oncology, 16:65.

[0095] In some embodiments, a first toxin-conjugated antibody may target a surface protein of an HSC. In some embodiments, a first toxin-conjugated antibody may target selectively bind a surface protein of an HSC. (and / or selectively bind to) GPI-80 (glycosylphosphatidylinositol-anchored protein 80, also known as VNN2). In some embodiments, a first toxin-conjugated antibody may target (or selectively bind to) a receptor tyrosine kinase (e.g., c-Kit (CD117)). In some embodiments, a first toxin-conjugated antibody may target (or selectively bind to) aAttorney Docket No. 9865.247. WOglycosylphosphatidylinositol (GPI)-anchored protein, e.g. a GPI-80 (VNN2). In some embodiments, a first toxin-conjugated antibody may target (or selectively bind to) both a receptor tyrosine kinase (e.g., c-Kit) and a (GPI)-anchored protein (e.g., GPI-80). In some embodiments, a first toxin-conjugated antibody may target (or selectively bind to) a receptor tyrosine kinase (e.g., c-Kit) and a (GPI)-anchored protein (e.g., GPI-80). In some embodiments, a first toxin-conjugated antibody may be target (or selectively bind to) both a receptor tyrosine kinase (e.g., c-Kit) and a GPI-anchored protein (e.g., GPI-80), wherein the first toxin-conjugated antibody targets exclusively to only one of the two targets. For example, a first toxin-conjugated antibody can be target (or selectively bind to) c-Kit and GPI-80, but once c-Kit (or GPI-80) is targeted (or selectively bound) the GPI-80 (or c-Kit, respectively) can no longer be targeted (or selectively bound). In some embodiments, when a first toxin-conjugated antibody targets (or selectively binds to) a GPI-anchored protein (e.g., GPI-80), the first toxin-conjugated antibody may target both lymphocytes and myeloid cells.

[0096] In some embodiments, second toxin-conjugated antibody may target (or selectively bind to) a cell surface protein of a lymphocyte. In some embodiments, a second toxin-conjugated antibody may target (or selectively bind to) a cell surface protein of a myeloid cell. In some embodiments, second toxin-conjugated antibody may target (or selectively bind to) CD56. In some embodiments, a second toxin-conjugated antibody may target (or selectively bind to) CD335. In some embodiments, a second toxin-conjugated antibody may target (or selectively bind to) IL2RG. In some embodiments, a second toxin-conjugated antibody may target (e.g., selectively bind to) CD56, CD335, and / or IL2RG.

[0097] The present invention is explained below in further detail in the following nonlimiting examples.EXAMPLESExample 1

[0098] Using non-genotoxic conditioning to dissect the role that niche availability, host HSC competition, and fetal immunity play in the engraftment of adult donor HSC following lUTx.

[0099] During development, definitive hematopoiesis begins in the AGM region, migrates to the fetal liver, and finally to the BM, where it persists throughout life. This shift in the site of hematopoiesis is thought to involve the large-scale migration of HSC via the circulation. It was initially assumed that HSC could piggyback upon this naturally occurring migration and seed the newly developing niches, thereby facilitating donor HSC engraftment after lUTx. However, it is now recognized that the fetal hematopoietic system is incredibly competitive and has aAttorney Docket No. 9865.247. WOproliferative advantage over the transplanted adult donor HSC, and that far less nascent niche “space” is likely available to donor HSC than was originally presumed. In addition to endogenous HSC competition, current evidence supports the presence of more robust fetal immunity than was initially appreciated during the period of so-called “pre-immunity.” As such, haploidentical (maternal or paternal) or allogeneic donor HSC also face immune barriers that need to be overcome to achieve curative engraftment post-IUTx. This conclusion is supported by the fact that the only clinical cures following lUTx were in X-SCID.

[0100] For postnatal HSC transplantation, the issues of space, host competition, and immune rejection of donor HSC are all addressed by administering genotoxic conditioning to the recipient in the form of ionizing radiation or chemotherapeutics like busulfan. However, such approaches are not viable options for tackling the barriers to engraftment in the fetus.

[0101] Recently, researchers have been exploring the use of toxin-conjugated antibodies to CD117 / c-kit as a means of depleting murine and xenografted human HSC in vivo and thereby achieving non-genotoxic conditioning. This method results in the highly specific and highly efficient depletion of both short-term and long-term engrafting HSC, while sparing more mature hematopoietic lineages. Moreover, we have developed and optimized an in-house method for producing and purifying a recombinant version of the type I ribosome-inactivating protein toxin saporin and its subsequent bioconjugation to streptavidin, enabling production of a cell-specific immunotoxin by simple linkage to a biotinylated antibody to the desired target protein, as shown in FIG. 1. Although previous research has administered toxin-conjugated c-kit antibody to NSG mice in utero, such research simultaneously performed lUTx with human HSC, as the anti-mouse c-kit antibody did not cross-react with the transplanted human cells. Past research established the feasibility of using antibody-toxin conjugates for safe in utero conditioning, but engraftment levels were still relatively low, likely due to the inability of the xenogeneic host to fully support the transplanted human HSC. Thus, the critical question of whether such an approach would work in the more clinically relevant setting of lUTx with haploidentical or allogeneic HSC (rather than xenogeneic HSC) remains unanswered.

[0102] Moreover, subsequent studies showed that optimal HSC depletion by c-kit immunotoxin only occurs at ~5 days post-administration. As such, past research did not accurately test the full potential of non-genotoxic conditioning in the context of lUTx. Since murine gestation is only 19-20 days and lUTx cannot be performed until day 14.5, such studies are not possible in mice. In addition, prior work with both sheep and human bone marrow has shown that c-kit is not expressed by most primitive HSC during fetal development; rather, mostAttorney Docket No. 9865.247. WOc-kit+ cells in the early fetal bone marrow co-express Stro-1, indicating they are niche-derived, as shown in FIG. 2.

[0103] It has been shown that the fraction of human CD34+ cells enriched for LTE are c-kitdim. As such, c-kit alone may not be the ideal marker to target for depleting host HSC in the setting of lUTx. Instead, glycosylphosphatidylinositol-anchored protein GPI-80 (VNN2) has been shown as a surface marker whose expression distinguishes self-renewing HSC and allows tracking of HSC in multiple developmental hematopoietic niches during human fetal ontogeny. Importantly, research shown the presence of GPI-80+ HSC in the human fetal bone marrow as early as 15 gestational weeks, suggesting GPI-80 would be an ideal target to deplete endogenous fetal HSC prior to lUTx.

[0104] A head-to-head comparison was thus performed between saporin-conjugated antibodies to c-kit and to GPI-80 for their ability to clear niche “space” and knock down host competition to enable efficient donor allogeneic (sheep) HSC engraftment following lUTx in fetal sheep. Antibodies are commercially available that recognize sheep c-kit (NP_001295523.1) and GPI-80 (XP_004011388.4) (>85% identity to human), simplifying production of the saporin-conjugated immunotoxins to these markers.

[0105] Of note is that curative lUTx for X-SCID was achieved in the absence of conditioning, demonstrating enough “space” is available within the nascent bone marrow niches to allow donor HSC to engraft and persist in sufficient numbers to repopulate the lymphoid compartment of the recipient. What was absent in these patients, however, was any form of fetal immune barrier, arguing that the fetal immune system, while not “mature,” can still mount a sufficient response to preclude donor HSC engraftment. Early NK cells in the fetal liver that express adult levels of alloreactive receptors have been suggested as causing this phenomenon, suggesting that NK cells may pose a barrier to engraftment of transplanted cells as early as the end of the first trimester in humans. Previous research has shown that depleting NK cells from the fetus enhances allogeneic cell engraftment following lUTx. However, in these studies the antibody used to deplete murine NK cells targeted an antigen not found on human or sheep NK cells (NK1.1), and so these findings have thus not been reproduced in a large animal lUTx model.

[0106] The present study uses a saporin-conjugated antibody to CD56 / NCAM-1 (commercial anti-hCD56 antibodies tested cross-react with sheep), the antigen expressed on human and sheep NK cells at the same maturation states as NK1.1 in mice. While depletion of NK cells enhances donor HSC engraftment following lUTx in mice, the only time complete repopulation has been observed following lUTx (in mice or humans) has been in cases when the recipientAttorney Docket No. 9865.247. WOfetus harbored an immunodeficiency. Moreover, it has been reported that the cotransplantation of mesenchymal stem cells (MSC) with HSC enhances the level of donor HSC following lUTx, and that this enhancement occurs irrespective of whether the MSC are transplanted 3 days prior to the HSC or simultaneously, as shown in FIG. 3. While it could be argued that transplanting the MSC 3 days prior to the HSC “primed” the bone marrow niches to receive the transplanted HSC, the ability of the MSC to exert the same effect on engraftment when concurrently transplanted suggested that the MSC could be exerting their effect on engraftment by modulating the immune system of the fetal recipient.

[0107] To test this theory, we transplanted fetal sheep with MSC alone and examined the levels of T cells in the recipients. As seen in FIG. 4, transplantation of MSC alone markedly reduced the levels of CD4+, CD8+, and CD4+CD8+ T cells in the recipient when compared to non-IUTx age-matched controls. To determine whether the presence of functional immunity at the time of lUTx is the factor limiting donor HSC engraftment, we tested whether transient induction of an “X-SCID-like” state at the time of lUTx, via administration of a saporin-conjugated antibody to IL2RG, is sufficient to enable high-level allogeneic HSC engraftment.FIG. 5 shows an overview of the Experimental Design.

[0108] Experiment 1.1: Testing the impact of depleting endogenous HSC prior to lUTx using saporin-conjugated antibodies to: a) c-kit and b) GPI-80 on the engraftment of allogeneic sheep HSC.

[0109] To prepare the saporin-conjugated immunotoxins to CD117 / c-kit and to GPI-80, we used biotinylated antibody to c-kit or GPI-80 combined at a 1:1 molar ratio with an in-house-developed streptavidin-saporin product. C-kit- or GPI-80-saporin immunotoxin was delivered intravenously via the umbilical vein by percutaneous injection under continuous ultrasound (US) visualization at a dose of 0.5mg / kg into fetal sheep at 55-57 gestational days (term: 145 days), with an additional group of recipients receiving no immunotoxin serving as a control (n=16 fetuses / group). Allogeneic adult sheep bone marrow-derived CD34+ HSC was isolated using magnetic bead-sorting and an antibody we developed that is specific to sheep CD34. HSC was transduced with a SIN lentivector encoding tdTomato (genetic fusion of two copies of dTomato specifically designed for low aggregation, that behaves like a monomer and fluoresces > 3x brighter than GFP; CLONTECH® / TAKARA®) under a ubiquitous promoter (PGK, EF1α, UbiC) to enable tracking and quantitation of engraftment levels following sheep-to-sheep lUTx. At 5 days after immunotoxin administration (the time of maximal depletion of endogenous / host hematopoiesis), transduced HSC were transplanted into these fetal sheep using a clinically employed dose of 108cells / kg in a 0.5ml volume, intraperitoneally (IP) byAttorney Docket No. 9865.247. WOpercutaneous injection, under continuous US visualization, comparing pre- and post-IUTx fetal heart rate to ensure well-being.

[0110] Prior work over the past 25 years using the sheep model to study the engraftment and differentiation of a variety of human and sheep HSC subsets has demonstrated that the levels of donor cell engraftment observed in the peripheral blood (PB) and bone marrow at 60 days post-IUTx are an accurate early indicator of the levels of long-term engraftment that will ultimately be achieved. For this reason, to ascertain what impact conditioning with c-kit or GPI-80 immunotoxin has on engraftment following lUTx, half of the lUTx recipients (n=8 fetuses / group) were euthanized, and PB and bone marrow collected and analyzed by flow cytometry for tdTomato and for expression of a panel of lineage-specific antigens to: 1) quantify the levels of donor cell engraftment and 2) assess the multilineage nature of the engraftment. The other half of the lUTx recipients (n=8 fetuses / group) will be allowed to complete gestation and be bom for long-term analysis. PB and bone marrow samples will be collected from these animals at monthly intervals following birth for up to 6 months of age, by which time donor cells are lost if long-term engrafting / repopulating HSC (LTE-HSC) have not engrafted following lUTx. These samples were also analyzed by flow cytometry for tdTomato and for expression of a panel of lineage-specific antigens, as just described for the euthanized recipients. tdTomato+ cells FACS-sorted from the monthly bone marrow samples were also evaluated for clonogenic potential using a high-throughput luminescence-based HALO® (HEMOGENIX®). Aliquots of sorted tdTomato+ cells were cryopreserved for future serial repopulation studies to verify the successfully engraftment of LTE-HSC in these preconditioned lUTx recipients. Engraftment levels determined by flow cytometry were confirmed in both PB and bone marrow by qPCR for the tdTomato reporter in the lentivector.

[0111] Experiment 1.2: Testing the impact of depleting host NK cells alone or NK, T, and B cells prior to lUTx using saporin-conjugated antibodies to: a) CD56 and b) IL2RG, respectively, on allogeneic HSC engraftment. To test the role fetal NK, T, and B cells play in limiting allogeneic HSC engraftment post-IUTx and determine how significant an impact available niche “space” vs. immune barriers exert, we performed studies that are similar to those detailed in Experiment 1.1, but administered saporin-conjugated antibody (0.5mg / kg) to either CD56 or to IL2RG, to deplete NK cells alone or NK, T, and B cells, respectively, 5 days prior to lUTx with tdTomato+ allogeneic CD34+ sheep HSC (108cells / kg) prior to lUTx (n=16 fetuses / group). As above, half of the lUTx recipients (n=8 fetuses / group) were euthanized at 60 days post-IUTx for FACS analysis of the levels and multilineage nature of donor cell engraftment, while the remaining half (n=8 fetuses / group) were allowed to be born for long-Attorney Docket No. 9865.247. WOterm analysis of multilineage engraftment on monthly PB and bone marrow samples and HALO® (HEMOGENIX®) assays on FACS-sorted tdTomato+ cells from the monthly bone marrow samples. Engraftment levels determined by flow cytometry in both PB and bone marrow were confirmed using qPCR with primers specific to the tdTomato reporter gene within the lentiviral vector. We cryopreserved aliquots of sorted tdTomato+ cells for future serial repopulation studies to verify the successful engraftment of LTE-HSC in these immune-depleted lUTx recipients. To reduce the animals required for these studies, the group of recipients in Experiment 1.1 that received no immunotoxin served as control (n=16 fetuses / group).

[0112] Experiment 1.3: Testing the impact of combined depletion of both endogenous HSC and immune cells prior to lUTx on allogeneic HSC engraftment. Having determined, in Experiments 1.1 and 1.2 the optimal immunotoxin to deplete endogenous HSC (c-kit or GPL 80) and nascent immune cells (CD56 or IL2RG) within the fetus that hinder engraftment of allogeneic HSC following lUTx, we tested the hypothesis that combining these two immunotoxins will maximize HSC engraftment post-IUTx and enable us to achieve our target of 20-25% donor cell engraftment. To accomplish this objective, we administered saporin-conjugated antibody to the HSC marker that yields optimal depletion of endogenous HSC in Experiment 1.1 and to the immune marker that yields maximal HSC engraftment in Experiment 1.2 (0.5mg / kg for each antibody-conjugate) 5 days prior to lUTx with tdTomato+ allogeneic CD34+ sheep HSC (108cells / kg; n=16 fetuses). As in the preceding Experiments, half of the lUTx recipients (n=8 fetuses / group) were euthanized at 60 days post-IUTx for FACS analysis of the levels and multilineage nature of donor cell engraftment, while the remaining half (n=8 fetuses / group) were used for long- term analysis (after birth) of multilineage engraftment on monthly PB and bone marrow samples and HALO® (HEMOGENIX®) assays on FACS-sorted tdTomato+ cells from the monthly BM samples. Engraftment levels in both PB and bone marrow determined by flow cytometry was confirmed using qPCR with primers specific to the tdTomato reporter. As before, we cryopreserved aliquots of sorted tdTomato+ cells for future serial repopulation studies. To reduce the number of animals required for these studies, the group of recipients from Experiment 1.1 that received no immunotoxin served as a control (n=16 fetuses / group). Results obtained with this combined conditioning regimen were compared to that obtained with each of the immunotoxins alone to determine the degree of improvement depleting both endogenous HSC (i.e., host competition and “space”) and immunity has on donor HSC engraftment.Attorney Docket No. 9865.247. WO

[0113] Outcomes of Experiment 1. We surprisingly identified a safe and efficient non-genotoxic conditioning regimen that depletes the host HSC in utero, reducing competition faced by the transplanted HSC and freeing “space” within the bone marrow for high level engraftment. Using also an immunotoxin targeting GPI-80 was more effective than targeting c-kit alone. Based on clinical experience with lUTx and our preliminary data on T cell levels within the fetus at the time of lUTx and the ability of co-transplanted MSC to reduce these levels and enhance HSC engraftment, immunotoxin targeting IL2RG was more effective than targeting NK cells via CD56.

[0114] An important consideration when designing immunotoxins for depleting specific cell types is the turnover rate of the protein being targeted. Proteins with slow turnover do not yield optimal internalization of the antibody-saporin conjugate and therefore do not mediate very efficient elimination of the cells expressing the targeted antigen. Importantly, postnatal studies demonstrated the efficacy of immunotoxin-mediated conditioning targeting c-kit. The half-life of IL2RG on the cell surface is only about 1 hr, making it an ideal target for saporin-conjugated antibody -based conditioning. While antibodies to CD56 have not been tested for depleting NK cells, CD56-targeted drug conjugates are currently being used to target small cell lung cancer, attesting to the efficacy of CD56-targeted antibodies for delivering toxins that are internalized. No data concerning the half-life of GPI-80 had been discovered prior to this study, and our data confirm efficient uptake and killing with the anti-GPI-80-saporin conjugate in vitro.

[0115] Experient 2: Performing competitive repopulation studies to determine the impact of the phenotype and functionality of the donor HSC on the levels of engraftment following lUTx.

[0116] The small size of the fetal recipient (limiting the volume of donor cells that can be injected) and the low frequency of HSC within the adult bone marrow necessitate that lUTx be performed with populations of enriched / purified HSC. For this reason, all lUTx procedures that have been performed clinically with adult bone marrow-derived HSC have used CD34+, a marker that is present on HSC from most species. However, there is a large amount of data demonstrating that the most primitive HSC reside within the fraction that is devoid of CD34, especially for fetal sources. Indeed, FIG. 6 shows that during fetal ontogeny, the majority of the primitive HSC leaving the fetal liver to seed the nascent bone marrow do not express CD34. Instead, most of these HSC express the surface marker CD166 / ALCAM. This finding is of note, as prior work by the showed that CD 166 is a functional marker of both murine and human (adult) HSC as well as cellular components of the murine bone marrow niche, and further demonstrated that murine CD 166+ HSC were markedly enriched for bone marrow-repopulating cells. Moreover, it was shown that homophilic interactions between CD 166 onAttorney Docket No. 9865.247. WOthe surface of HSC and that on the surface of cells within the bone marrow niche contribute to a more robust hematopoietic outcome. Given its ability to identify HSC with enhanced repopulating potential and its presence on the HSC present within the bone marrow at the time of lUTx, we tested whether selecting donor HSC for expression of CD 166, rather than CD34, will enhance the ability of the graft to compete with endogenous host (fetal) HSC and yield higher donor HSC engraftment post-IUTx.

[0117] In addition to selecting a subset of HSC with enhanced repopulating potential, a second strategy used to improve donor cell competitiveness was to enhance their homing to the developing fetal bone marrow niches and their ability to respond to niche-derived hematopoietic factors. CD26 is an enzyme expressed on HSC 88 that cleaves the aminoterminal dipeptide from SDF-1 / CXCL12, producing an inactive form that prevents chemotaxis of HSC along SDF-1 gradients and alters the ability of HSC to respond to critical hematopoietic cytokines. FIGs. 7A-C show data on levels of donor HSC engraftment within the perivascular regions of the bone marrow following lUTx correlated with the levels of SDF-1 being expressed by co-transplanted CD146+ niche cells that engrafted within the vasculature, attesting to the importance of this chemotactic gradient following lUTx.

[0118] The role of CD26 in hematopoiesis has been reported as modulating CD26 activity to enhance engraftment of cord blood-derived HSC to sufficient levels to enable single cord units to be used in adolescent and adult patients. Based on highly promising studies in mice, the CD26 inhibitor sitagliptin was tested in a clinical trial for engraftment of cord blood-derived HSC 90. While the drug was safe and well-tolerated and the data suggested an improved engraftment level, CD26 inhibition mediated by sitagliptin is of short duration (hours). Interestingly, in the first publication investigating the impact of CD26 disruption on HSC engraftment in mice 88, researchers observed that using donor HSC from CD26- / - mice yielded far higher engraftment levels of HSC with serial-repopulating ability than did transient drug-mediated CD26 activity blockade on wild- type donor HSC, providing compelling evidence that more durable inhibition of CD26 on the donor HSC would exert a more pronounced effect on engraftment. The data from the sitagliptin clinical trial supports this conclusion, as does the modest reported improvement in engraftment using transient drug-based inhibition of CD26 in a murine model of lUTx. These prior preclinical and clinical data provide the basis for our hypothesis that long-lasting knockdown of CD26 on donor HSC via shRNA will markedly enhance donor LTE-HSC engraftment levels following lUTx. An overview of Experiment 2 appears in FIG. 8.Attorney Docket No. 9865.247. WO

[0119] Experimental Approach: To test the impact of the phenotype / functionality of the donor HSC and the impact durably inhibiting CD26 has on the levels of HSC engraftment post-lUTx, adult allogeneic sheep bone marrow-derived HSC were isolated based on expression of CD34 or CD 166 using magnetic bead-sorting. We then transduced the isolated HSC populations with a “bipartite” 162 lentivector encoding GFP for CD34+ HSC or tdTomato for CD 166+ HSC, and either a scramble (control) shRNA or an shRNA targeting CD26. Three CD26-targeted shRNAs were designed and tested via transient transfection, and the one yielding maximal knockdown was incorporated into the lentivector using the HORIZON® SMARTVECTOR® system. Following transduction, HSC were flow sorted on a BECKMANCOULTER ASTRIOS EQ® to obtain pure GFP+ and tdTomato+ populations of CD34+ and CD166+ HSC, respectively. Successful CD26 knockdown was confirmed by FACS. The CD34+ and CD 166+ HSC transduced with the lentivector encoding a scramble shRNA was mixed in a 1:1 ratio, as will the CD34+ and CD 166+ HSC transduced with the lentivector encoding CD26-targeted shRNA. Each of these two mixed populations of transduced HSC were transplanted into a separate cohort of fetal sheep (n=16 / group) that have been conditioned using the regimen that yields the highest levels of donor cell engraftment in Experiment 1, herein. HSC were transplanted using a clinically employed total cell dose of 108cells / kg, IP by percutaneous injection, under continuous US visualization, comparing pre- and post-IUTx fetal heart rate to ensure well-being. As in Experiment 1, half of the lUTx recipients (n=8 fetuses / group) were euthanized at 60 days post-IUTx for FACS analysis of the levels and multilineage nature of donor cell engraftment, while the remaining half (n=8 fetuses / group) were used for long-term analysis (after birth) of multilineage engraftment on monthly PB and bone marrow samples, and HALO® (HEMOGENIX®) assays on FACS-sorted GFP+ and tdTomato+ cells from the monthly bone marrow samples. Engraftment levels in both PB and bone marrow determined by flow cytometry were confirmed using qPCR with primers specific to the GFP and tdTomato reporter. We also cryopreserved aliquots of sorted GFP+ and tdTomato+ cells for future serial repopulation studies. By performing parallel studies in which CD34+ vs CD166+ HSC expressing a control / scramble shRNA are forced to compete for engraftment following lUTx and studies in which CD34+ vs CD 166+ HSC expressing a CD26-targeted shRNA are forced to compete for engraftment following lUTx, we were able to simultaneously determine the impact of HSC phenotype / function (CD34+ vs. CD166+) and of CD26 inhibition on the levels of donor HSC engraftment achieved following lUTx. The use of competitive repopulation studies rigorously tested the LTE potential of each HSC populationAttorney Docket No. 9865.247. WOwhile simultaneously removing any uncertainties caused by animal-to-animal variability, as the two test HSC populations were interrogated within the same animal.

[0120] Outcomes of Experiment 2. CD34+ HSC engrafted at low levels following lUTx. CD34+ HSC depleted of CD26 expression engrafted at significantly higher levels without any alterations to their long-term engrafting and differentiation abilities. Transplanting HSC selected for CD 166 has not been attempted in the setting of lUTx, but in the present study these cells yielded higher levels of engraftment further enhanced when CD26 expression is knocked down. Since colony-forming potential has been shown to be a good predictor of in vivo engraftment, we performed CFU methylcellulose assays on an aliquot of each HSC population being transplanted. Flow cytometry was performed to verify the purity of the magnetic bead- sorted CD34+ and CD 166+ HSC. The use of a lentivector encoding an shRNA to CD26 resulted in long-lasting knockdown of CD26 without any untoward effects on HSC function.

[0121] Experiment 3: Testing the therapeutic efficacy of lUTx for treating / curing SCD by performing lUTx in the SCD sheep. Extensive analyses of human SCD patients who have undergone HSC transplantation (postnatally) suggest that levels of myeloid engraftment of >20% are needed to achieve phenotypic correction of SCD. As such, SCD represents an ideal disease to have tested the therapeutic potential of lUTx. Having determined the optimal parameters for conditioning and the ideal donor HSC phenotype to consistently obtain donor HSC engraftment levels >20% following lUTx in normal / healthy fetal sheep in Experiments 1 and 2, we tested the ability of our optimized lUTx approach to achieve long-term correction in SCD sheep using the clinical and laboratory tests detailed in Experiment 3.1 to assess whether lUTx is curative or the degree to which it improves the clinical phenotype. To accomplish this objective, we generate SCD animals via SCNT and embryo transfer, and by breeding of affected animals (and heterozygotes, if needed) once they have reached sexual maturity. At 35-45 days of gestation (term: 145 days), the resultant fetuses were screened by amniocentesis for the SCD mutation using a PCR-based RFLP. We then administered saporin-conjugated antibody to the HSC marker and / or immune marker that yields optimal depletion of endogenous HSC in Experiment 1 (0.5mg / kg for each antibody-conjugate) 5 days prior to performing lUTx (n=5 SCD fetuses) with tdTomato+ allogeneic sheep HSC a clinically employed dose of 108cells / kg in a 0.5ml volume, intraperitoneally (IP) by percutaneous injection under continuous ultrasound visualization. The outcome of Experiment 2 determined whether the donor HSC was selected for CD34 and / or CD 166 and whether they are depleted of CD26 expression prior to lUTx. Near term, ewes were placed under observation and were either induced into labor for natural delivery, or lambs were delivered by C-section. All lUTxAttorney Docket No. 9865.247. WOrecipients were used for long-term analysis (after birth) of correction of the SCD phenotype (using the assays detailed in Experiment 3.1) and for multilineage engraftment on monthly PB and bone marrow samples and HALO® (HEMOGENIX®) assays on FACS-sorted tdTomato+ cells from the monthly bone marrow samples. Engraftment levels in both PB and bone marrow determined by flow cytometry will be confirmed using qPCR with primers specific to the tdTomato reporter.

[0122] Animal Group Size and Gender '. 8 animals (fetuses) were used per experimental group throughout the proposed studies to ensure differences observed were statistically significant and reproducible. An animal number / group of 5 was determined by power analyses using G*POWER® v3.1.9.2, to achieve significance, assuming a type I error of 5%, an effect size of 5, a STDEV of 3% within each group and a desired power of 80%. This conclusion was then verified using the “resource equation method,” which predicted that an “E” value of >20 is achieved with 5 animals / group, confirming no additional statistical value would be obtained with increased group size. Nevertheless, we erred on the conservative side and used 8 animals / group, as this was the first time the proposed immunotoxin-based conditioning and the knockdown of CD26 are being tested in a large animal model of lUTx. Data suggest levels of engraftment after lUTx is not gender-related, and as such female and male fetuses were used. Given the effort required to generate SCD animals, the lUTx studies used sample sizes of n=5.

[0123] Domestic female sheep (O. aries), 2 to 5 years of age, served as recipients for somatic cell nuclear transfer (SCNT) embryo implantations. Embryo recipients were of various breeds including Suffolk, Suffolk crosses, Western white-faced and Polypay. Cloned offspring were produced from genetically modified sheep fetal fibroblasts originated from American Romney sheep fetuses. For embryo transfer procedure, sheep were anesthetized with a cocktail consisting of Xylazine (0.05 mg / kg body weight) and acepromazine (0.03 mg / kg bodyweight) with a final induction of ketamine (6 mg / kg body weight, VEDCO®) given intravenously. A 4-5” midline incision was made to allow the uterus and ovaries to be exteriorized. The embryos were transferred into the ostium of the infundibulum via a tomcat catheter and deposited at least 3.5 cm into the oviduct. The tract was then gently placed back into the abdominal cavity and the incision was closed. Pregnancies were diagnosed by ultrasonography on day 40±3 of gestation.

[0124] Generation of a Sheep Model of Sickle Cell Disease (SCD): A sheep model of Sickle Cell Disease (SCD) was generated. Twenty embryo transfers were performed in the fall of Year 1 using SCD male fibroblast cells (previously established) as nuclear donors for somatic cell nuclear transfer (SCNT). Forty female sheep per year were used as recipients for SCNT embryoAttorney Docket No. 9865.247. WOtransfers in Years 2 and 3 of the project. For detailed model characterization, both male and female SCD lambs were produced. As the severity of SCD in sheep was unknown, lambs with monoallelic mutations (SCD carriers) were also generated. The goal was to produce four homozygous and four heterozygous (carrier) SCD lambs per gender via SCNT (16 lambs in total).

[0125] Experimental details: Twenty recipients per group were used for embryo transfers (20 in Year 1 and 40 in Years 2-3). Twenty additional recipients were included in the Year 3 in case cloning efficiency with SCD cells was lower than expected. Fifteen ±5 SCNT embryos were transferred per recipient. The estimated pregnancy rate at Day 40 of gestation was 30-35%, resulting in approximately 6-7 pregnancies per group. The delivery rate was 20-25%, averaging 1.2 lambs per recipient. This approach yielded an expected 4-6 lambs per group. These were conservative assumptions, as the typical SCNT lambing rate ranged between 25% and 35%, resulting in the production of over 50 genetically modified cloned sheep over the past four years. Additional embryo transfers (n = 20) were performed in Year 3 if necessary. In total, 80 sheep recipients were used for embryo transfers in this aim (20 per group). In the event of reduced cloning efficiency, the total number of recipients in the study was increased to 100 animals.Example 2: Investigating Novel Non-Genotoxic Conditioning Approaches for In Utero Hematopoietic Stem Cell Transplantation

[0126] Hematopoietic stem cell (HSC) transplantation in utero (lUTx) has been performed safely for decades in humans and successfully cured X-SCID, with all the patient's T lymphocytes arising from the donor. However, for reasons that are not well understood, successful HSC engraftment after lUTx has only been achieved in X-SCID fetal recipients. We hypothesize that depletion of endogenous HSC and / or immune cells is necessary to achieve therapeutic levels of donor HSC engraftment following lUTx.

[0127] Here, we investigated which antibody-drug conjugates (ADC) would be able to create niche availability, eliminate host HSC competition, and thwart nascent immunity, thereby enabling engraftment of adult donor HSC at therapeutic levels following lUTx. Using a fetal sheep model, we used immunohistochemistry to identify which immune cells and / or HSC populations are present in the fetal bone marrow (BM) at the time of lUTx. Flow cytometric analysis of sheep BM was then used to identify antibodies suitable for use as ADC to target HSC (CD45+GPI-80) and immune cells (CD45+IL2RG). Antibody internalization using a pH-dependent dye, and cell death assays using ADC and saporin as a toxin were also performed toAttorney Docket No. 9865.247. WOconfirm internalization and determine optimal concentration of the respective immunotoxins. Sheep fetuses at 55-57 gestational days were conditioned using GPI-80- or IL2RG-saporin ADC and were then transplanted 5-7 days later with human adult donor CD34+HSC, at a dose of 107cells / kg. Engraftment levels of human donor cells were determined in peripheral blood (PB) and bone marrow (BM) samples at 60 days post-IUTx and at various time points postbirth. In all conditioned lUTx recipients, levels of human HSC(CD34+), T(CD3+), (CD7), B(CD10+)(CD20+), and myeloid (CD123+) cells in PB and BM were determined by flow cytometry after exclusion of sheep CD45+ cells. At 60 days post-IUTx, levels of human cells in GPI-80-conditioned recipients were: CD34+ (0.5±0.02%;5.8±0.5%), CD3+ (5.72±0.4%;14.93±1.0%), CD7+ (7.62±3.6%;13.4±0.73%), CD10 (3.49±2.0%;9.49±4.9%), CD20+ (3.88±0.9%;11.18±2.1%), and CD123+ (6.48±0.9%; 6.2±2.1%), in PB and BM, respectively. Similar levels of human T, B, and myeloid cells were observed in IL2RG-conditioned fetuses: CD34+(0.68%;0.95), (CD3+(3.13%;14.2%), CD7+(2.78%;0.15%), CD10 (5.05%, 0.59%), CD20+(8.63%,14.4%), and CD123+(6.12%; 3.2%), in PB and BM, respectively. Clonogenic assays in GPI-80- and IL2RG-conditioned lUTx recipients demonstrated the presence of human CFU-GEMM, BFU-E, CFU-GM, and CFU-E, therefore establishing the ability of the engrafted human HSC to proliferate and differentiate into multiple hematopoietic lineages. Total human donor cells in PB of both GPI-80- and IL2RG-conditioned animals at birth (4 months after lUTx) decreased considerably to around 2%. However, at 6 months after lUTx, the levels of human CD3+, CD7+, CD20+, and CD123+ cells in the PB of GPI-80 conditioned animals increased to 5.57%, 5.4%, 2.86%, and 1.12%, respectively, while engraftment in IL2RG-conditioned animals remained low. In conclusion, when compared with historical controls of HSC-transplanted fetuses without conditioning, the depletion of recipient HSC or immune cells prior to lUTx each appears to promote successful short-term donor HSC engraftment, while the selective depletion of recipient HSC seems to be critical to achieve long-term engraftment of donor HSC.Example 3: Enhancing Donor Cell Engraftment in Prenatal Hematopoeitic Stem Cell Transplantation with Novel Antibody-Drug Conjugates

[0128] In-utero hematopoietic stem cell (HSC) transplantation (lUTx) has been safely performed in humans for decades, but clinical efficacy has only been achieved in fetal recipients with X-SCID. We hypothesize that lUTx can deliver curative levels of HSC engraftment in other genetic disorders after the selective depletion of highly proliferative fetal HSC and / or immune cells. We developed novel antibody-drug conjugates (ADCs) using theAttorney Docket No. 9865.247. WOribosomal toxin, saporin, to selectively deplete NK cells expressing CD335, immune cells expressing IL2RG, and fetal HSCs expressing GPI-80. Using a pH-dependent fluorescent reporter and cytotoxic assays, we showed that the maximum internalization and endocytosis of antibodies targeting GPI-80, IL2RG, and CD335 occurred at 6, 18, and 12 hours (FIGs. 9A-C), and that 150nM of GPI-80, IL2RG, and CD335 killed bone marrow (BM) or peripheral blood (PB) mononuclear cells at 50%, 40%, and 10%, respectively (FIGs. 10A-C). To evaluate the safety profile and compare GPI-80-, IL2RG- and CD335-ADCs' off-target effects, such as targeting of other cell populations besides hematopoietic cells, with other more studied ADCs such as c-kit, immunohistochemistry was performed on fetal sheep tissues. Fetal kidney, brain, and testes contained 10±3.5%, 25±7.0% and 10±3.5% c-kit positive cells, respectively while GPI-80+, IL2RG+, and CD335* cells were negligible in these organs (FIGs. 11A-F; FIG. 12).However, IL2RG expression was identified in specific areas of microvascular endothelial cells within the kidney and testes (FIGs. 11 A-F; FIG. 12). In contrast, in the bone marrow (BM), levels of GPI-80+, IL2RG+, CD335+, and c-kit+ cells were 46.6±1.9%, 47.9±1.6%, 6.5±1.6%, and 17.7±2.8%, respectively (FIGs. 13D; FIG. 14). In the thymus, 20*0.9% of cells were IL2RG+ (FIG. 13B; FIG. 14). Thus, GPI-80, c-Kit, IL2RG, and CD335 are primarily expressed in the hematopoietic organs (FIGs. 13A-D; FIG. 14).

[0129] Having confirmed ADC's functionality and safety profile, human adult BM-derived CD34+ HSC (n=2) at a dose of 108 / kg were injected (IP) into fetal sheep at 62-65 gestation days, 1 week after administration of 50ug of GPI-80-ADC (n=4), IL2RG-ADC (n=5), CD335-AD (n=4), or saline (n=3) (non-ADC control). Prior to lUTx, flow cytometric analysis showed that 97±0.9% of the transplanted cells were CD45+CD34+, and of these, 34±3% and 39±5% were CD38“ and CD133+, respectively. CFU-assays confirmed their robust clonogenic potential. Engraftment levels of human donor cells were determined in PB (FIG. 15A) and BM (FIG. 15B) at 60 days post-IUTx by flow cytometry and Digital PCR. The latter was performed using primers specific to the human GAPDH and RPLPO genes. Copies / ml of human GAPDH and RPLPO genes were: for GPI-80 (BM:305±132; 539±50; PB:110±46; 162±48), for IL2RG (BM:18±5; 53±16; PB:10±3; 44±7), for CD335 (BM:80±10; 64±10; PB:21±2; 43±6), and for No- ADC (BM:15±3; 34±19; PB:3±1; 13±3) (FIGs. 15A-B), Flow cytometric analysis of fetal sheep BM showed that the engraftment in BM and PB was multilineage with the presence of lymphoid and myeloid cells. Human CD45 engraftment was significantly higher in both GPI-80 (BM:19.4 ± 1.8%, PB:11.8 ± 2.1%) and CD335 (BM:16.4± 4.7%, PB:4.3 ± 1.2%) groups when compared with IL2RG (BM:7.5 ± 2.3%, PB.3.4 * 1.0%) and No-ADC (BM:2.3 ± 1.1%, PB:8.0 * 2.6%) (FIGs. 15C-D). All ADCs also significantly increased the engraftment ofAttorney Docket No. 9865.247. WOhuman CD34+HSC when compared to the No-ADC control (BM:0.4±0.2%); (GPI-80: BM:3.4±0.6%, PB:2.0±0.6%); (IL2RG: BM:2.1±0.8% PB:1.8±0.7%); (CD335 5.6±2.2%, PB:0.8 ± 0.2%) (FIG. 15C). GPI-80 also significantly increased the engraftment of CD45+CD33+myeloid cells when compared to No-ADC control: (BM: GPI-80:39.3±9.8%, PB: 19.4±5.0%), (BM: IL2RG:4.8±0.8%, PB:8.8±3.0%), (BM: CD335:7.3±2.5%, PB:15.8±5.1%), (BM: No-ADC:7.1±6.7%, PB:7.7±3.5%) (FIG. 15D).

[0130] Additionally, to determine whether conditioning caused hematologic or metabolic alterations, complete blood cell counts (CBC, including white blood cells (K / pL) and hematocrit (%); FIGs. 16A-B) and liver enzymes (AST and ALT; FIGs. 16C-D) were also evaluated. All ADC groups displayed similar CBC counts and AST levels compared to the No- ADC group. However, ALT was elevated in the IL2RG and CD335 ADC groups when compared to the No-ADC group. Histopathological analyses of fetal tissues in transplanted animals demonstrated no evidence of ADC-associated toxicity in any of the examined tissues.

[0131] In summary', while all selected ADCs displayed minimal off-target effects and selective ablation of endogenous HSCs, GPI-80 conditioning tends to skew engraftment toward the myeloid lineage, while IL2RG and CD335 conditioning tend to skew engraftment toward the B lymphocyte lineage, with GPI-80 thus far being the optimal drug candidate to achieve therapeutic levels of HSC engraftment after lUTx.Example 4: Lipid Nanopartide-Based Agent for Prenatal Non-Genotoxic Conditioning (NGC)

[0132] Prior work has shown that by encapsulating modified in vitro transcribed mRNA (mmRNA) encoding only the catalytic domain (CD) of pseudomonas exotoxin A (PEA), without PEA’ s binding and translocation domains, one retains potent intracellular toxicity upon mRNA translationfl]. Delivering only the CD via mRNA in lipid nanoparticles (LNPs) may also eliminate any potential for toxin leakage and “bystander” toxicity[l],

[0133] While LNPs are the most advanced delivery vehicle for mRNA[2-7], the clinical efficacy of their cargo is often limited by phagocytosis of the LNPs by cells of the mononuclear phagocytic system (MPS)[8, 9], which triggers inflammation. MPS cells express the phagocytosis regulator, SIRPa on their surface[10, 11], Kupffer cells (KCs), MPS cells residing in the liver, account for up to 15% of the total liver cellular mass and express SIRPa[12, 13], Also, while in the circulation, LNPs bind to serum proteins (e.g., ApoE) to form a protein corona, driving their uptake by hepatocytes via the LDL receptor (LDL-R)[14-16], As a result of these combined mechanisms, the majority of systemically administered LNPs accumulateAttorney Docket No. 9865.247. WOin the liver and not in the desired target cells. The anti -phagocytic counterpart of SIRPa is the cell surface protein CD47, which acts as a ubiquitous “don’t eat me” signalflO, 17-20], We will “cloak” the anti-GPI-80 “decorated” LNPs (loaded with mmRNA encoding PEA-CD) with CD47 peptide[8] to prevent their uptake by MPS cell s[8], and we will formulate the LNPs with enhanced PEGylation to mask ApoE binding sites[2] and swap cholesterol with synthetic glutamate- cholesterol (GA-Chol)

[0021] , both of which have been shown to dramatically reduce LDL-R-mediated hepatocyte uptake of LNPs. By incorporating CD47 and optimizing the composition of the LNPs, we expect to markedly enhance their uptake by HSC and minimize potential toxicity due to non-specific uptake.

[0134] We hypothesize that by incorporating CD47 “cloaking” and optimizing the chemical composition of anti-GPI-80 “decorated” LNPs (loaded with toxin mRNA), we will markedly enhance their uptake by HSC and minimize potential toxicity due to non-specific uptake, thereby creating a novel, safe agent for prenatal NGC.

[0135] We will create anti-GPI-80 “decorated” LNPs, loaded with mRNA encoding PEA-CD to mediate prenatal NGC. To prevent their uptake by phagocytes, we will “cloak” the LNPs with CD47 [8], We will also employ enhanced PEGylation to mask ApoE binding sites and replace cholesterol with synthetic GA-Chol, both of which dramatically reduce LDL-R-mediated hepatocyte uptake of LNPs[2, 21, 25, 26],

[0136] Approach: Generation of anti-GPI-80 “decorated” LNPs with unique chemical composition to avoid binding of serum ApoE with resultant uptake by hepatocyte LDL-R, and CD47 “cloaking” to avoid phagocytosis. To achieve efficient and specific delivery to fetal hematopoietic stem cells (HSC), we will begin by fabricating LNPs based on “lipid 10,” a composition previously shown to exhibit very efficient delivery to hematopoietic cells

[0027] , but replace cholesterol with synthetic GA-Chol

[0021] and formulate the LNPs with enhanced PEGylation to mask ApoE binding sites[2, 25, 26], as both of these modifications have been shown to markedly reduce LDL-R-mediated uptake of LNPs by hepatocytes. A custom CD47 peptide containing the sequence of GNYTCEVTELSREGKTVIELK (SEQ ID NO:1) will be synthesized, and micelles containing the CD47 peptide will be incorporated into the LNPs via post-insertion mixing as in[8, 28], These customized LNPs will then be conjugated to a GPI-80 antibody using standard maleimide-thiol chemistry [29-32] to create HSC-targeted LNPs (HSC-tLNPs). After preparation, conjugation, and purification; the size, uniformity, and zeta potential of the HSC-tLNPs will be evaluated, and the uniformity of the HSC-tLNPs before and after conjugation will be confirmed by TEM as in

[0033] .Attorney Docket No. 9865.247. WO

[0137] References:1. Granot-Matok, Y., et al., Lipid nanoparticles-loaded with toxin mRNA represents a new strategy for the treatment of solid tumors. Theranostics, 2023. 13(11): p. 3497-3508. 2. Kim, M., et al., Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver. Sci Adv, 2021. 7(9).3. Polack, F. P., et al., Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med, 2020. 383(27): p. 2603-2615.4. Rosenblum, D., et al., CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy. Sci Adv, 2020. 6(47).5. Jayaraman, M., et al., Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew Chem Int Ed Engl, 2012. 51(34): p. 8529-33.6. Rietwyk, S. and D. Peer, Next-Generation Lipids in RNA Interference Therapeutics.ACS Nano, 2017. 11(8): p. 7572-7586.7. Semple, S. C., et al., Rational design of cationic lipids for siRNA delivery. Nat Biotechnol, 2010. 28(2): p. 172-6.8. Papp, T. E., et al., CD47 peptide-cloaked lipid nanoparticles promote cell-specific mRNA delivery. Mol Ther, 2025. 33(7): p. 3195-3208.9. Kiaie, S. H., et al., Recent advances in mRNA-LNP therapeutics: immunological and pharmacological aspects. J Nanobiotechnology, 2022. 20(1): p. 276.10. Oronsky, B., et al., Just eat it: A review of CD47 and SIRP-alpha antagonism. Semin Oncol, 2020. 47(2-3): p. 117-124.11. Duan, X., et al., Dichotomous role of CD47-SIRPalpha axis: Leveraging opposing functions in phagocytic resistance and transport assistance for enhanced nanocarrier biocompatibility and tumor penetration. Acta Pharm Sin B, 2026. 16(3): p. 1605-1624.12. Bouwens, L., et al., Quantitation, tissue distribution and proliferation kinetics of Kupffer cells in normal rat liver. Hepatology, 1986. 6(4): p. 718-22.13. Deng, Z., etal., The nuclear factor ID3 endows macrophages with a potent anti-tumour activity. Nature, 2024. 626(8000): p. 864-873.14. Chu, R., et al., Lipid nanoparticles as the drug carrier for targeted therapy of hepatic disorders. J Mater Chem B, 2024. 12(20): p. 4759-4784.15. Kockx, M., M. Traini, and L. Kritharides, Cell-specific production, secretion, and function of apolipoprotein E. J Mol Med (Berl), 2018. 96(5): p. 361-371.Attorney Docket No. 9865.247. WO16. Paunovska, K., et al., The Extent to Which Lipid Nanoparticles Require Apolipoprotein E and Low-Density Lipoprotein Receptor for Delivery Changes with Ionizable Lipid Structure. Nano Lett, 2022. 22(24): p. 10025-10033.17. Brown, E. J. and W. A. Frazier, Integrin-associated protein (CD47) and its ligands. Trends Cell Biol, 2001. 11(3): p. 130-5.18. Oldenborg, P. A., et al., Role of CD47 as a marker of self on red blood cells. Science, 2000. 288(5473): p. 2051-4.19. Rodriguez, P. L., et al., Minimal " Self1peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles. Science, 2013. 339(6122): p. 971-5.20. Jaiswal, S., et al., CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell, 2009. 138(2): p. 271-85.21. Sahel, D. K., et al., Improved localized mRNA delivery using lipid nanoparticles with a novel synthetic cholesterol derivative. RSC Pharm, 2026. 3(1): p. 187-197.22. Somu Naidu, G., et al., Ionizable Lipids with Optimized Linkers Enable Lung-Specific, Lipid Nanoparticle-Mediated mRNA Delivery for Treatment of Metastatic Lung Tumors. ACS Nano, 2025. 19(6): p. 6571-6587.23. Yang, F. and T. A. Scott, Advances in lipid nanoparticles delivering genetic medicines for solid cancers. Mol Ther Nucleic Acids, 2026. 37(1): p. 102838.24. Zhou, R., et al., Lipid Nanoparticle-Encapsulated mRNAs Encoding Tumor-Specific Toxin Proteins Selectively Target Cancer Cells and Stimulate T-cell Infiltration. Cancer Res, 2025. 85(19): p. 3706-3716.25. Mui, B. L., et al., Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles. Molecular Therapy - Nucleic Acids, 2013. 2: p. el39.26. Ryals, R. C., et al., The effects of PEGylation on LNP based mRNA delivery to the eye. PLOS ONE, 2020. 15(10): p. e0241006.27. Ramishetti, S., et al., A Combinatorial Library of Lipid Nanoparticles for RNA Delivery to Leukocytes. Adv Mater, 2020. 32(12): p. el906128.28. Ishida, T., D. L. Iden, and T. M. Allen, A combinatorial approach to producing sterically stabilized (Stealth) immunoliposomal drugs. FEBS Lett, 1999. 460(1): p. 129-33.29. Breda, L., et al., In vivo hematopoietic stem cell modification by mRNA delivery. Science, 2023. 381(6656): p. 436-443.30. Breda, L., et al., Erratum for the Research Article " In vivo hematopoietic stem cell modification by mRNA delivery" by L. Breda et al. Science, 2025. 388(6751): p. eadz0744.Attorney Docket No. 9865.247. WO31. Parhiz, H., et al., PEC AM-1 directed re-targeting of exogenous mRNA providing two orders of magnitude enhancement of vascular delivery and expression in lungs independent of apolipoprotein E-mediated uptake. J Control Release, 2018. 291: p. 106-115.32. Rurik, J. G., et al., CAR T cells produced in vivo to treat cardiac injury. Science, 2022.375(6576): p. 91-96.33. Tarab-Ravski, D., et al., Delivery of Therapeutic RNA to the Bone Marrow in Multiple Myeloma Using CD38-Targeted Lipid Nanoparticles. Adv Sci (Weinh), 2023. 10(21): p. e2301377.

[0138] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

Attorney Docket No. 9865.

247. WOWHAT IS CLAIMED IS:

1. A method of enhancing engraftment of donor hematopoietic stem cells (dHSC) in a recipient in utero subject, comprising:(a) depleting hematopoietic stem cells of the recipient in utero subject (rHSC) by administering to the recipient in utero subject a toxin-conjugated antibody targeting the rHSC;(b) depleting lymphocytes (e.g., NK, T and / or B cells) of the recipient in utero subject by administering a toxin-conjugated antibody targeting the lymphocytes; or(c) performing both (a) and (b).

2. The method of claim 1, wherein the toxin-conjugated antibody of (a) and / or (b) comprises an antibody covalently or non-covalently linked to a nanoparticle (e.g., a lipid nanoparticle).

3. The method of claim 2, wherein the nanoparticle is a lipid nanoparticle comprising mRNA encoding the toxin.

4. The method of any one of claims 1-3, wherein the toxin of (a) and / or (b) is saporin, Pseudomonas exotoxin A (PE), Diphtheria toxin (DT), ricin A chain, gelonin, bouganin, abrin A chain, colicin E3, alpha-sarcin, and / or BLF1 (Burkholderia lethal factor 1).

5. The method of any one of claims 1-3, wherein the toxin of (a) and / or (b) is alpha-Amanitin, MMAE (monomethyl auristatin E), or the catalytic domain of Pseudomonas exotoxin A (PE).

6. The method of any of one of claims 1-5, wherein the toxin-conjugated antibody of (a) targets (e.g., selectively binds to) GPI-80 (glycosylphosphatidylinositol-anchored protein 80, also known as VNN2).

7. The method of any one of claims 1-5, wherein the toxin-conjugated antibody of (b) targets (e.g., selectively binds to) CD56, CD335 and / or IL2RG.

8. The method of claim 7, wherein the conjugated antibody of (b) targets IL2RG.Attorney Docket No. 9865.

247. WO9. The method of any one of claims 1-8, wherein the toxin-conjugated antibody of (a) and / or (b) also targets and depletes myeloid cells of the recipient in utero subject.

10. The method of any one of claims 1-9, wherein the method further comprises engrafting of the donor hematopoietic stem cells (dHSC) in the recipient in utero subject.

11. The method of claim 10, further comprising, prior to the engrafting of the dHSC in the recipient in utero subject, enhancing dHSC homing to a desired hematopoietic niche of the recipient in utero subject, thereby further enhancing engraftment of dHSC.

12. The method of claim 11, wherein enhancing dHSC homing comprises, prior to the engrafting of the dHSC in the recipient in utero subject, reducing expression of an enzyme (e.g., a cell-surface protease, such as peptidyl-peptidase) of the dHSC to increase responsiveness of the dHSC to chemokine signaling directing the dHSC to the desired hematopoietic niche.

13. The method of claim 11 or claim 12, wherein the desired hematopoietic niche is bone marrow and / or liver of the recipient in utero recipient.

14. The method of any one of claims 1-13, further comprising, prior to the engrafting of the dHSC in the recipient in utero subject, enriching for dHSCs that express CD 166.

15. A composition useful for enhancing engraftment of donor hematopoietic stem cell (dHSC) in a recipient in utero subject, comprising:a first toxin-conjugated antibody that targets an extracellular domain of a hematopoietic stem cell that is endogenous to the recipient in utero subject (rHSC); and / or, a second toxin-conjugated antibody that targets an extracellular domain of a lymphocyte that is endogenous to the recipient in utero subject,wherein the composition comprises a pharmaceutically acceptable carrier.

16. The composition of claim 15, wherein the first toxin-conjugated antibody and / or the second toxin-conjugated antibody comprises an antibody covalently or non-covalently linked to a nanoparticle (e.g., a lipid nanoparticle).Attorney Docket No. 9865.

247. WO17. The composition of claim 16, wherein the nanoparticle is a lipid nanoparticle comprising mRNA encoding the toxin.

18. The composition of any one of claims 15-17, wherein the toxin of the first toxin-conjugated antibody and / or the second toxin-conjugated antibody is saporin, Pseudomonas exotoxin A (PE), Diphtheria toxin (DT), ricin A chain, gelonin, bouganin, abrin A chain, colicin E3, alpha-sarcin, alpha-Amanitin, MMAE (monomethyl auristatin E), and / or BLF1 (Burkholderia lethal factor 1).

19. The composition of any one of claims 15-17, wherein the toxin of the first toxin-conjugated antibody and / or the second toxin-conjugated antibody is alpha-Amanitin, MMAE (monomethyl auristatin E), or the catalytic domain of Pseudomonas exotoxin A (PE).

20. The composition of any one of claims 15-19, wherein the first toxin-conjugated antibody targets (e.g., selectively binds to) GPI-80 (glycosylphosphatidylinositol-anchored protein 80, also known as VNN2).

21. The composition of any one of claims 15-20, wherein the second toxin-conjugated antibody targets (e.g., selectively binds to) CD56, CD335, and / or IL2RG.

22. The composition of any one of claims 15-20, wherein the second toxin-conjugated antibody targets (e.g., selectively binds to) IL2RG.

23. The composition of any one of claims 15-22, wherein the first and / or second toxin-conjugated antibody also targets an extracellular domain of a myeloid cell that is endogenous to the recipient in utero subject.

24. Use of the composition of any one of claims 15-23 in a method of enhancing engraftment of donor hematopoietic stem cells (dHSC) in a recipient in utero subject, or for the manufacture of a medicament for enhancing engraftment of donor hematopoietic stem cells (dHSC) in a recipient in utero subject.

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