Tolerogenic dendritic cells as an adjunct to stem cell-derived b-cell replacement therapy for type 1 diabetes

Abstract

The present disclosure pertains to a method for treating Type 1 diabetes mellitus by using tolerogenic dendritic cells (tDCs) as an adjunct to stem cell-derived β-cell (SCislet) replacement therapy. The tDCs are genetically modified to induce immunotolerance and are administered in conjunction with SCislet autografts or allografts to enhance their survival and function, providing a potential cure or disease-modifying treatment for T1DM.

Description

USE OF TOLEROGENIC DENDRITIC CELLS AS AN ADJUNCT TO STEM CELL- DERIVED B-CELL REPLACEMENT THERAPY FOR TYPE 1 DIABETES MELLITUSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims priority to U.S. Provisional Application No. 63 / 662,932 filed on June 21, 2024, and 63 / 791,199 fded on April 18, 2025, each of which are incorporated herein by reference in their entirety.FIELD OF INVENTION

[0002] The present disclosure relates to the field of biopharmaceutics and cell therapy, particularly to methods and compositions for treating Type 1 diabetes mellitus (T1DM) through the use of tolerogenic dendritic cells (tDCs) as an adjunct to stem cell-derived [3-ccl 1 surrogate therapy.BACKGROUND

[0003] Type 1 diabetes mellitus (T1DM) is characterized by the autoimmune destruction of insulin-producing [3-cclls in the pancreas, necessitating exogenous insulin replacement therapy. Current therapeutic strategies, including islet transplantation, are limited by donor shortages, the risk of alloimmune and autoimmune rejection, and the lifelong immunosuppression requirements. Advances in stem cell-derived J3-cells (SCislets) offer potential for scalable and effective [3-cell surrogate therapy, yet challenges remain in ensuring their engraftment, maturation, and long-term function within the autoimmune milieu of T1DM patients.SUMMARY

[0004] The present disclosure describes a novel approach for enhancing the efficacy and longevity of SCislet allografts in T1DM therapy. The disclosed methods involve the use of recipient-derived and / or stem cell-sourced dendritic cells (DCs) that are rendered tolerogenic through genetic and molecular manipulations. These tDCs are designed to induce immunotolerance to the SCislet allografts, thereby mitigating alloimmune and / or autoimmune responses and promoting allograft or autograft survival and function. The tDCs may be administered systemically or locally in various formulations and timings relative to the SCislet allograft or autograft placement.

[0005] Some embodiments are directed to methods for treating Type 1 diabetes mellitus (T1DM) comprising: generating tolerogenic dendritic cells (tDCs) by downregulating expressionof genes involved in the adaptive immune response in dendritic cells (DCs); exposing said tDCs to antigens associated with stem cell-derived P-cells (SCislets) or to materials used for encapsulating SCislets to induce allograft or autograft-specific immunotolerance; and administering said tDCs to a subject in conjunction with placement of SCislet grafts to enhance survival and function of the SCislet allograft or autografts. In some embodiments, the genes involved in the adaptive immune response include CD40, CD80, and CD86. In some embodiments, the tDCs are generated by exposure to antisense oligonucleotides, short interfering ribonucleic acids (siRNAs), or through gene editing technologies. In some embodiments, the gene editing technology is CRISPR / Cas9. In some embodiments, the tDCs are derived from the recipient of the SCislet allograft or autografts. In some embodiments, the tDCs are derived from the same induced pluripotent human stem cell (ihPSC) source as the SCislets. In some embodiments, the tDCs are administered systemically. In some embodiments, the tDCs are administered locally at or around the SCislet allograft or autograft site. In some embodiments, the tDCs are administered before, during, or after the placement of the SCislet grafts. In some embodiments, the methods disclosed herein further comprise encapsulating the SCislet grafts prior to placement. In some embodiments, the encapsulating material is selected to promote host vascular invasion and vascularization of the SCislet allograft or autografts. In some embodiments, the methods disclosed herein further comprise a step of pre-exposing the tDCs to stress-induced antigens expressed by the SCislets during post-implantation maturation. In some embodiments, the tDCs are administered in a formulation designed to target dendritic cells. In some embodiments, the tDCs are administered in conjunction with microspheres containing oligonucleotides for in vivo generation of immunotolerant dendritic cells. In some embodiments, the methods disclosed herein further comprising administering an immunosuppressant. In some embodiments, the immunosuppressant is selected from tacrolimus, sirolimus, etanercept, thymoglobulin or any combination thereof. In some embodiments, the immunosuppressant is administered before, during, or after the administration of the tDCs and SCislet allograft or autografts. In some embodiments, the methods disclosed herein further comprise administering insulin therapy. In some embodiments, the insulin therapy is administered before, during, or after the administration of the tDCs and SCislet allograft or autografts. In some embodiments, the methods disclosed herein further comprise administering an anticoagulant. In some embodiments, the anticoagulant is heparin. In some embodiments, the anticoagulant is administered before, during, or after the administration of the tDCs and SCislet allograft or autografts. In some embodiments, the methods disclosed herein further comprise administering aspirin. In some embodiments, the aspirin is administered before, during, or after the administration of the tDCs and SCislet allograft or autografts.

[0006] Some embodiments are directed to pharmaceutical compositions comprising tolerogenic dendritic cells (tDCs) generated by the method of claim 1 for use in treating Type 1 diabetes mellitus (T1DM).

[0007] Some embodiments are directed to kits for treating Type 1 diabetes mellitus (T1DM) comprising: a first component containing tolerogenic dendritic cells (tDCs) generated by the methods disclosed herein; and a second component containing stem cell-derived J3-cells (SCislets) or materials for encapsulating SCislets. In some embodiments, the kits described herein further comprising instructions for administering the tDCs and SCislets to a subject in a manner that enhances the survival and function of the SCislet allograft or autografts.

[0008] Some embodiments are directed to methods for inducing immunotolerance in a subject with Type 1 diabetes mellitus (T1DM) comprising: administering a therapeutically effective amount of tolerogenic dendritic cells (tDCs) generated by the methods disclosed herein; and concurrently administering stem cell-derived [ -cclls (SCislets) to the subject to promote insulin independence. In some embodiments, the tDCs and SCislets are administered in a manner that mitigates alloimmune and / or autoimmune responses in the subject. In some embodiments, the tDCs are administered in a dosage and timing regimen optimized based on the subject's immune profile and the characteristics of the SCislet allograft or autografts.BRIEF DESCRIPTION OF THE FIGURES

[0009] Figure 1 depicts the effect of antisense-oligodeoxynucleotide (AS-ODN) modified DCs and exposure to freshly isolated intact allogeneic-to-the-recipient islet-derived lysate on preserving allograft insulin secretory function and normoglycemia in streptozotocin diabetic mice.

[0010] Figure 2 depicts peritransplant management for islet allotransplantation.DETAILED DESCRIPTION

[0011] Before compounds, compositions and methods are described in detail, it is to be understood that this disclosure is not limited to the particular processes, compositions, or methodologies described, as these may vary. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope of the disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the disclosure, the preferred methods, devices, and materials are now described.

[0012] The present disclosure provides detailed embodiments of a method for treating T1DM using tDCs in conjunction with SCislet allografts or autografts. The tDCs are generated by downregulating gene expression involved in the adaptive immune response, such as CD40, CD80, and CD86, using antisense oligonucleotides, siRNAs, or gene editing technologies like CRISPR / Cas9. Additionally, tDCs may be exposed to SCislet antigens or encapsulating materials to enhance allograft or autograft-specific tolerance. The administration of tDCs can be tailored to the SCislet manufacturing process and may involve various routes and timings to optimize the therapeutic outcome.

[0013] Type 1 diabetes mellitus (T1DM) results from the autoimmune impairment, injury and destruction of native insulin producing J3-cells in the islets of Langerhans of the pancreas resulting in the need for exogenous insulin replacement therapy to treat, but not cure, T1DM. Dendritic cells (DCs) are endogenous antigen presenting cells that detect and ingest native and foreign matter within the body and then transport and present it to the immune system as either native or foreign to induce, respectively, protective immunotolerance or adaptive inflammatory responses. The autoimmune destruction of [3-cells in T1DM results from false identification of native [3-cells as foreign by the immune system. Applicant has developed methods for the artificial creation of tolerogenic DCs (tDCs) and their autologous local injection proximal to immune tissue draining the pancreas to halt the progressive autoimmune [3-cell destruction in newly and recently diagnosed T1DM, thereby eliminating or delaying the need for exogenous insulin. This curative or disease-modifying immunotherapy would necessarily apply only to new onset or very early- stage T1DM wherein significant (3-cell functional reserve is retained. Curative therapy for more advanced T1DM would require (3-cell replacement involving transplantation of cadaveric donor- derived whole pancreas or islets of Langerhans isolated therefrom. Shortage of organ donors plus the need for multiple donors to yield sufficient mass of isolated islets combined with necessary lifelong immunosuppression to prevent alloimmune and autoimmune destruction has severely limited the practicality of this approach, although the broad application of tDCs to address both the allo-immunity and autoimmunity of islet transplantation for T1DM has been widely considered (Sun 2012, Made Ion 2020, Pathak 2021). An alternate approach to cadaveric organ donor-sourced islets is to artificially manufacture well-functioning insulin-secreting human pancreatic islets or insulin secreting [3-cell surrogates from an induced human pluripotent stem cell (ihPSC) source (SCislets) (Hogrebe 2023, Karimova 2022). SCislets could be genetically engineered (Gerace 2023, Parent 2021, Sackett 2022, Sintov 2022) and / or encapsulated (Alagpulinsa 2019, Bochenek 2019, Dolgin 2016, Keymeulen 2023) to better evade auto- and / or allo-immunity and other stressors following implantation in patients with T1DM. Despite greatadvances in the molecular and cell biology of human SCislets (Balboa 2022), their practical application as a scalable, safe, and effective P-cell surrogate replacement therapy for T1DM remains elusive, with significant challenges related to their manufacture, engraftment and maturation, and to the allo- and autoimmune host environment in recipients with T1DM (Balboa 2022, Hogrebe 2023, Karimova 2022, Keymeulen 2023). These challenges could be abrogated in part by novel adaptation of tDC-based methodologies that have been well-studied and described for allo- and xeno-transplantation of donor-derived mature islet allografts (Ali 2000, Bass 2014, Bhatt 2012, Cai 2017, Hughes 2020, Ozeri 2012, Pathak 2021, Pothoven 2010, Rastellini 1995, Stepkowski 2006, Sun 2012, Thomas 2013) but not for SCislet allografts (Hogrebe 2023, Karimova 2022) that present unique set of challenges and opportunities. SCislets require additional post-implant functional maturation to attain the robust oxidative glucose metabolism and glucose-linked insulin secretion characteristic of mature islets (Balboa 2022, Hogrebe 2023, Karimova 2022). Persistence of genomic and epigenetic modifications and / or cell phenotypic instability post-implantation combined with manufactured hypo-immunogenicity in ihPSC- derived SCislets could constitute a long-term safety risk to recipients (Gerace 2023, Kabakchieva 2023). The present invention describes the use of recipient-derived and / or stem cell sourced DCs (Cai 2017) that (a) are rendered generally tolerogenic (versus immunogenic) by in vitro (Giannoukakis 2011) or in vivo (Di Caro 2012) exposure to antisense oligonucleotides (ASO’s) or short interfering ribonucleic acids (siRNA’s) or Crisp R / Cas9 or similar gene editing technologies that induce the downregulation of the genetic expression of DC molecules necessary for induction of adaptive T-cell immune response (intDCs), and (b) may also be further rendered allograft or autograft-specific tolerogenic by subsequent pre-exposure to components or molecules characteristic of the SCislets and / or SCislet precursors and / or graft encapsulating material (pextDCs). The intDCs and / or pextDCs would be administered to SCislet recipients systemically (e.g. intravenously) and / or locally (at or around the SCislet graft site and / or in the SCislet graft lymphatic drainage field) before, contemporaneous with, and / or following SCislet graft placement. The tDCs would be intended to dampen the combined acute immunologic and metabolic stressors uniquely faced by SCislets during their critical post-engrafting maturation (Balboa 2022, Hogrebe 2023, Karimova 2022) and encapsulation and vascularization (Bochenek 2018, Karimova 2022, Keymeulen 2023, Liang 2023), and prolong their survival and function thereafter in an immune-intolerant milieu.

[0014] The invention is the utilization of artificially created tDCs as an adjunct to the implantation of allograft or autograft SCislets to treat or cure T1DM. The tDCs are produced by inducing the suppression of the expression of key molecules involved in induction of the T-celladaptive immune response (intDCs) such as CD40, CD80, and CD86 using either specific antisense or interfering oligonucleotide (ASO’s or siRNAs) targeting the primary transcripts of the genes, or using CrispR / Cas9 or similar gene editing technologies, and by further rendering DCs specifically immunotolerant to the graft by pre-exposing them to antigens and other molecular constituents of the allograft or autograft SCislet (pextDCs). The DCs to be converted to tDCs by these methods could be sourced either directly from the SCislet allograft or autograft recipient (r-DCs) (by leukapheresis) or from the same ihPSC source from which the SCislets were derived (sc-DCs) (Li 2014) whose immuno-compatibility would further advance the likelihood and potency of graft immunotolerance. Thus, four classes of tDCs are to be considered: r-intDCs, sc-intDCs, r-pextDCs, and sc-pextDCs.

[0015] Each of the four classes of tDCs could be administered to the allograft recipient systemically (e.g. intravenously) or locally (within the SCislet allograft or autograft itself, within or adherent to the SCislet allograft or autograft encapsulating structure, into the adjacent tissue or into the same anatomical compartment as the SCislet allograft or autograft , and / or into tissue in the same lymphatic drainage field as the SCislet allograft or autograft). These four classes of tDCs could be administered before, concurrent with, and / or following allograft placement to acutely pre-treat or concurrently treat and / or chronically treat the allograft or autograft SCislet and / or recipient thereby to better initiate, protect, preserve, promote and sustain allograft or autograft survival and function including the early initiation and later preservation of robust glucose-stimulated insulin secretion to induce and sustain insulin independence in patients with T1DM.

[0016] The exact method of tDC toleration and administration could vary according to the process and procedure of SCislet manufacture, encapsulation and placement into the recipient. For example, in cases where allograft-specific tolerogenicity is to be provided to the recipient prior to or with allograft placement, sc-intDCs or r-intDCs would be pre-exposed by co-culture with the developing or developed SCislets to induce an allograft-specific tolerogenic state and then separated and harvested from the SCislet culture by microgravity conditions (Rutzky 2002) or cell sorting procedures. Alternatively, r-tDCs or sc-tDCs could be pre-exposed to SCislet antigens by incubation with SCislet lysates or pre-exposed to synthetically produced SCislet proteins or peptides derived from sequencing the relative proteins of interest (including but not limited to proteins from the human leukocyte antigen (HLA) loci: HLA locus [class I, class II HLA]). Preexposure of tDCs by co-culture, lysate incubation or synthetic proteins / peptides could be conducted relevant to SCislets purposefully exposed to metabolic stress to specifically extend and condition tolerogenic protection to SCislet antigens expressed or over-expressed uniquely duringthe critical post-implantation phase when adequate vascularization (Hogrebe 2023), maturation and mature glucose-induced oxidative metabolism is not yet achieved (Balboa 2022). In cases where SCislet allografts will be encapsulated with protective materials, tDCs also could be preexposed and pre-tolerized to the protective encapsulating material (Keymeulen 2023, Vegas 2016b, Wang 2021) with or without the presence of anticipated adherent extracellular matrix proteins (Leifer 2017, Wang 2022, Zu 2019) to better preserve SCislet function and mitigate the fibrotic foreign body response (Leifer 2017, Vegas 2016a).

[0017] Under circumstances where autologous SCislets are derived from the recipient’s ihPSC’s (i.e. a SCislet autograft), the method of tDC toleration and administration would vary according to the process and procedure of autologous SCislet manufacture, encapsulation and placement into the recipient. As in the case of SCislet allograft described above, sc-tDCs would be derived from the same recipient ihPSC source as the SCislets (Li 2014), or r-tDC’s would be derived via a separate leukapheresis from the recipient’s peripheral blood monocytes (PBMC’s) (Giannoukakis 2011). As described above for allograft-specific tolerogenicity, in cases where autograft-specific tolerogenicity is to be provided to the recipient prior to or with autograft placement, sc-intDCs or r-intDCs would be pre-exposed by co-culture with the developing or developed SCislets to induce an autograft-specific tolerogenic state, and then separated and harvested from the SCislet culture by microgravity conditions (Rutzky 2002) or cell sorting procedures. Alternatively, r-tDCs or sc-tDCs could be pre-exposed to autograft SCislet antigens by incubation with SCislet lysates or pre-exposed to synthetically produced SCislet proteins or peptides derived from sequencing the relative proteins of interest with respect to islet autoimmunity in T1DM. Pre-exposure of tDCs by co-culture, lysate incubation or synthetic proteins / peptides could be conducted relevant to SCislets purposefully exposed to metabolic stress to specifically extend and condition tolerogenic protection to SCislet antigens expressed or overexpressed uniquely during the critical post-implantation phase when adequate vascularization (Hogrebe 2023), maturation and mature glucose-induced oxidative metabolism is not yet achieved (Balboa 2022). In cases where SCislet autografts will be encapsulated with protective materials, tDCs also could be pre-exposed and pre-tolerized to the protective encapsulating material (Keymeulen 2023, Vegas 2016b, Wang 2021) with or without the presence of anticipated adherent extracellular matrix proteins (Leifer 2017, Wang 2022, Zu 2019) to better preserve SCislet function and mitigate the fibrotic foreign body response (Leifer 2017, Vegas 2016a).

[0018] General tolerogenic induction by oligonucleotide-mediated downregulation of DC genes necessary for co -stimulatory surface proteins (Castenmiller 2021) such as CD40, CD80 and CD86 that activate the immune response (Phillips 2019) could be delivered to r-DCs either ex vivoor in vivo (Di Caro 2012, Giannoukakis 2011, Phillips 2008, Phillips 2019). sc-DCs would be rendered tolerogenic ex vivo by ASO or siRNA oligonucleotides (Giannoukakis 2011) or other gene editing techniques targeting these same co-stimulatory surface proteins. r-DCs would be rendered tolerogenic ex vivo (Giannoukakis 2011) in a similar fashion and / or rendered tolerogenic in vivo by oligonucleotide methodology (Di Caro 2012, Phillips 2008). In both cases, the relevant oligonucleotides are packaged in specific microspheres that are designed specifically to be presented to and phagocytosed by DCs either ex vivo (US patent 9,724,365 B2) or in vivo (US patents 7,884,085, 7,964,574B2). For in vivo delivery, oligonucleotide-containing microspheres are injected within or surrounding the intended allograft implantation site and / or relevant lymphatic drainage field, where they render resident r-DCs tolerogenic.

[0019] The specific procedures for injection of ex vivo generated tDCs or oligonucleotide- containing microspheres for in vivo generation of intDCs would vary depending upon the location of and procedure for introducing the allograft or autograft SCislet into the recipient, and the extent to which the goal is to mitigate only local allo / auto-immunity or to also address more widespread systemic autoimmunity as well, and whether protective encapsulation is present. A relevant example would be the proposed localized allograft or autograft engraftment of SCislets into the highly vascularized and accessible tissue compartment beneath the abdominal anterior rectus sheath (Liang 2023), in which case ex vivo generated tDCs and / or oligonucleotide-containing microspheres for in vivo tDC induction would be injected into the same and / or adjacent tissue compartment as the allograft or autograft SCislet and / or into surrounding accessible regions within the relevant lymphatic drainage field. It is plausible that under some circumstances, both ex vivo and in vivo generated tDCs could be combined in the management of allograft or autograft SCislet allo- and / or autoimmunity. A second example would be the use of tDCs in combination with the subcutaneous implantation of allograft or autograft SCislets in the form of SC-derived pancreatic islet progenitor cells encased in a device designed to permit and promote host vascular invasion and vascularization (Keymeulen 2023, Ramzy 2021, Shapiro 2021). Either r-tDCs or sc-tDC’s could be generated ex vivo by either artificial induction alone or combined artificial induction plus pre-exposure. In induction plus pre-exposure cases, r-pextDCs or sc-pextDCs could be generated by pre-exposure to allograft or autograft SCislets plus constituents / components of the encapsulation device with or without expected adherent extracellular matrix proteins (Keselowsky 2017, Leifer 2017). The resultant ex-vivo generated r-pextDCs or sc-pextDCs could be preinjected or simultaneously injected either systemically (e.g. intravenously) or locally injected into the subcutaneous implantation site, or pre-placed within the to-be-implanted device prior to its placement. Alternatively, r-pextDCs could be generated in vivo by preparing the subcutaneousimplantation site by local injection of oligonucleotide-containing microspheres to render resident DC’s tolerogenic.

[0020] The use of donor-strain SC-derived tDCs has been described for solid organ allograft transplantation between allogeneic in-bred strains of mice (Cai 2017, Todorova 2020). The combined therapeutic use of tDCs with a syngeneic allograft or autograft that are both derived from the same SC source to protect and preserve post-transplant allo / auto graft function is novel, as is the furtherance of graft-specific immuno-protection afforded by pre-exposure of the SC- derived tDCs to the syngeneic SC-derived differentiated SCislet allograft or autograft or derivatives thereof prior to combined delivery to the transplant recipient. In the case of an SCisletallograft or autograft, pre-exposure could comprise co-culture of the SC-derived differentiated tDCs with SCislets, pre-exposure of the tDCs to SCislet lysates, and / or exposure of tDCs to specific synthetically produced proteins or peptide antigens found to be expressed by SCislets in culture and / or after intentionally subjected to metabolic stress, and / or other components of the allograft. As discussed above, depending upon the nature, location and composition of the islet allograft or autograft, the type and composition of encapsulation, and the extent of anticipated allo- and autoimmunity, the dose, location and cadence of delivery of sc- pextDCs would vary accordingly.

[0021] Each of the above-listed scenarios would be explored in preclinical models of T1DM, including the autoimmune T1DM NOD mouse model, the DP-BB rat model, as well as, when appropriate, strep tozotocin- induced diabetic non^human primate models, where local / anatomical aspects better reflect human allografting.

[0022] The ex vivo and in vivo production of r-intDCs by exposure to oligonucleotides targeting CD40, CD80 and CD86 has been published (Di Caro 2012, Giannoukakis 2011, Phillips 2008, Phillips 2019) and is the subject of several patents (US patents 9,724,365B2, 7,884,085B2, 7,964,574B2).

[0023] Embodiments of the disclosed method and system for treating Type 1 diabetes mellitus (T1DM) using tolerogenic dendritic cells (tDCs) in conjunction with stem cell-derived [:1- cell (SCislet) allografts are provided herein.

[0024] In one embodiment, the method includes generating tDCs by downregulating the expression of genes involved in the adaptive immune response in dendritic cells (DCs). This may be achieved through the use of antisense oligonucleotides, short interfering ribonucleic acids (siRNAs), or gene editing technologies such as CRISPR / Cas9. The genes targeted for downregulation may include, but are not limited to, CD40, CD80, and CD86.

[0025] In another embodiment, the generated tDCs are exposed to antigens associated with SCislets or to materials used for encapsulating SCislets. This exposure is designed to induce allograft-specific immunotolerance, thereby preparing the tDCs to promote tolerance to the SCislet allografts once administered to a subject.

[0026] The subject may be a pediatric mammal. The subject may be a neonatal mammal. The subject may be a geriatric mammal. The subject may be a pediatric mammal at risk of developing a condition (e.g. T1DM). The subject may be a pediatric mammal with a condition (e.g. T1DM). The treatment method may be preferred for a pediatric mammal. In some instances, composition of injections, timing of injections, amount of injections, anatomical location of injections may be altered to accommodate a pediatric mammal. In some instances, composition of injections, timing of injections, amount of injections, anatomical location of injections may be altered to accommodate physically smaller or physically larger patients.

[0027] A pediatric mammal may be a human. A pediatric mammal may be a mouse or nonhuman primate. A pediatric mammal may be a human that is less than 18 years of age. A pediatric mammal may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 years old. A pediatric mammal may be an infant, such as a child of less than about 1 year of age. A pediatric mammal may be a young child, such as between about 1 year and about 2 years of age, between about 1 year and about 3 years of age, or between about 1 year and about 5 years of age. A pediatric mammal may be a child, such as between about 6 years and 10 years of age, between about 6 years of age and about 12 years of age. A pediatric mammal may be between about 11 years and 13 years of age. A pediatric mammal may be between about 11 years and 18 years of age. A pediatric mammal may be an adolescent, such as between about 13 years and 18 years of year.

[0028] The term “about,” as used herein and throughout the disclosure, generally refers to a range that may be 15% greater than or 15% less than the stated numerical value within the context of the particular usage, unless otherwise specified. For example, “about 10” would include a range from 8.5 to 11.5.

[0029] Treatment may be provided to the subject before clinical onset of the condition (e.g. type 1 diabetes). Treatment may be provided for about: 1 day, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years before onset of the condition. Treatment may be provided for more than about: 1 day, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, before clinical onset of the condition.Treatment may be provided for less than about: 1 day, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 10 years, 15 years, before clinical onset of the condition.

[0030] Treatment may be provided to the subject after clinical onset of the condition (e.g. type 1 diabetes). Clinical onset of T1DM may be the need for a subject to utilize insulin injections to regulate blood sugar levels. In pediatric patients, a blood sugar level below 70 mg / dl can be considered low and, in some instances, can be characterized by symptoms such as, e.g. sweating, hunger and / or shakiness. In pediatric patients, a blood sugar level above 200 mg / dl can be considered high and can be characterized, in some instances, by low energy, stomachaches, and / or difficulty breathing. In pediatric patients, about 70 to about 120 mg / dl blood sugar level is considered normal. In pediatric patients, about 120 to about 200 mg / dl blood sugar level is considered outside the normal range, but it can be within the goal or target range for pediatric patients trying to maintain blood sugar levels. For pediatric patients aged about 12 years and older, maintaining a blood sugar level from about 70 to about 150 mg / dl can be a goal (e.g. for pediatric patients with diabetes). For pediatric patients aged about five years of age to about eleven years of age, maintaining a blood sugar level from about 70 to about 180 mg / dl can be a goal (e.g. for pediatric patients with diabetes). For pediatric patients aged about five years of age or younger, maintaining a blood sugar level from about 80 to about 200 mg / dl can be a goal (e.g. for pediatric patients with diabetes). One skilled in the art will recognize that these ranges are standard guidelines and, e.g., individual target ranges may vary based on a patient's age, body size, development, and the like.

[0031] Clinical onset of T1DM may be hyperglycemia. Clinical onset of T1DM may be the inability for a subject to regulate blood glucose levels. Clinical onset of T1DM may be inflammation of the pancreas. Clinical onset of T1DM may be pancreatic beta cell autoimmunity. Clinical onset of T1DM may be partial destruction of pancreatic beta cell mass. Destruction of pancreatic beta cell mass may be inflamed tissue, expansion of fibrotic legions, cellular apoptosis, cellular necrosis, cellular loss of function (e.g. inability to produce insulin, reduced insulin production). Clinical onset of T1DM maybe about: 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% destruction of pancreatic beta cell mass. Clinical onset of T1DM may be more than about: 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% destruction of pancreatic beta cell mass. Clinical onset of T1DM may be less than about: 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% destruction of pancreatic beta cell mass. Clinical onset of T1DM may be complete destruction ofpancreatic beta cell mass. Clinical onset of type 1 diabetes may include the onset of one or more symptoms of T1DM such as blurred vision, nausea, hyperglycemia, fatigue, weakness, muscle cramps, peripheral neuropathy, retinopathy, nephropathy, ulcers, other symptoms, and combinations thereof.

[0032] Treatment after clinical onset may be about: 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or 20 years after clinical onset. Treatment after clinical onset may be more than about: 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 20 years after clinical onset. Treatment after clinical onset may be less than about: 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 20 years after clinical onset. In some instances, treatment may be provided within 5 years of clinical onset. In some instances, treatment may be provided before about 5 years of clinical onset. In some instances, treatment may begin within about 5 years of clinical onset. In some instances, treatment may begin within about 4 years of clinical onset. In some instances, treatment may begin within about 3 years of clinical onset. In some instances, treatment may begin within about 2 years of clinical onset. Treatment may be provided to the subject after two, three, four, or more consecutive measurements of nonfasting blood glucose level greater than a given concentration (e.g. 250 mg / dl, 300 mg / dl). Treatment may also include treating a mammal in a clinical trial.

[0033] In a further embodiment, the tDCs are administered to a subject in conjunction with the placement of SCislet autografts or allografts. The tDCs may be derived from the recipient of the SCislet autografts or allografts or from the same induced pluripotent human stem cell (ihPSC) source as the SCislets. Administration of the tDCs may be systemic or local, at or around the SCislet autografts or allograft site, and may occur before, during, or after the placement of the SCislet allografts.

[0034] In a further embodiment, a therapeutically effective amount of tDCs are administered to a subject in conjunction with the placement of a therapeutically effective amount of SCislet allografts. The therapeutically effective amount of tDCs may be derived from the recipient of thetherapeutically effective amount of SCislet autografts or allografts or from the same induced pluripotent human stem cell (ihPSC) source as the therapeutically effective amount of SCislets. Administration of the therapeutically effective amount of tDCs may be systemic or local, at or around the SCislet autografts or allograft site, and may occur before, during, or after the placement of the therapeutically effective amount of SCislet autografts or allografts.

[0035] A “therapeutically effective amount” of treatment may prevent, arrest, reverse, or reduce a condition (e.g. T1DM), may include preserving viability of remaining beta cell populations, reducing inflammation, reducing blood glucose levels to pre-diabetic levels, increasing suppressive B-cell populations, reducing T-cell populations, inducing retinoic acid (RA) production in dendritic cell (DC) populations, increasing tolerogenic DC populations, or combinations thereof.

[0036] In yet another embodiment, the SCislet autografts or allografts are encapsulated prior to placement. The encapsulating material may be selected to promote host vascular invasion and vascularization of the SCislet autografts or allografts. The tDCs may also be pre-exposed to stress- induced antigens expressed by the SCislets during post-implantation maturation to further enhance immunotolerance.

[0037] In some embodiments, the tDCs are administered in a formulation designed to specifically target the dendritic cells. Alternatively, the tDCs may be administered in conjunction with bioengineered particles that include but are not limited to microspheres, microparticles, nanoparticles, surface modified or formulated to contain molecules that alone or in combination induce a tolerogenic state to the DC containing oligonucleotides for in vivo generation of immuno tolerant dendritic cells.

[0038] In some embodiments, the therapeutically effective amount of tDCs are administered in a formulation designed to specifically target the dendritic cells. Alternatively, the therapeutically effective amount of tDCs may be administered in conjunction with bioengineered particles that include but are not limited to microspheres, microparticles, nanoparticles, surface modified or formulated to contain molecules that alone or in combination induce a tolerogenic state to the DC containing oligonucleotides for in vivo generation of immunotolerant dendritic cells.

[0039] In some embodiments, the methods disclosed herein further comprising administering an immunosuppressant. In some embodiments, the immunosuppressant is selected from tacrolimus, sirolimus, etanercept, thymoglobulin or any combination thereof. In some embodiments, the immunosuppressant is administered before, during, or after the administrationof the tDCs and SCislet autografts or allografts. In some embodiments, the methods disclosed herein further comprise administering insulin therapy. In some embodiments, the insulin therapy is administered before, during, or after the administration of the tDCs and SCislet autografts or allografts. In some embodiments, the methods disclosed herein further comprise administering an anticoagulant. In some embodiments, the anticoagulant is heparin. In some embodiments, the anticoagulant is administered before, during, or after the administration of the tDCs and SCislet autografts or allografts. In some embodiments, the methods disclosed herein further comprise administering aspirin. In some embodiments, the aspirin is administered before, during, or after the administration of the tDCs and SCislet autografts or allografts.

[0040] In some embodiments, the methods disclosed herein further comprising administering a therapeutically effective amount of an immunosuppressant. In some embodiments, the immunosuppressant is selected from tacrolimus, sirolimus, etanercept, thymoglobulin or any combination thereof. In some embodiments, the immunosuppressant is administered before, during, or after the administration of the tDCs and SCislet autografts or allografts. In some embodiments, the methods disclosed herein further comprise administering insulin therapy. In some embodiments, the methods disclosed herein further comprise administering a therapeutically effective amount of insulin. In some embodiments, the insulin therapy is administered before, during, or after the administration of the tDCs and SCislet autografts or allografts. In some embodiments, the methods disclosed herein further comprise administering a therapeutically effective amount of an anticoagulant. In some embodiments, the anticoagulant is heparin. In some embodiments, the anticoagulant is administered before, during, or after the administration of the tDCs and SCislet autografts or allografts. In some embodiments, the methods disclosed herein further comprise administering a therapeutically effective amount of aspirin. In some embodiments, the aspirin is administered before, during, or after the administration of the tDCs and SCislet autografts or allografts.

[0041] A pharmaceutical composition embodiment includes tDCs generated by the disclosed method for use in treating T1DM. Additionally, a kit embodiment for treating T1DM may comprise a first component containing the generated tDCs and a second component containing SCislets or materials for encapsulating SCislets. The kit may further include instructions for administering the tDCs and SCislets to enhance the survival and function of the SCislet autografts or allografts.

[0042] A pharmaceutical composition embodiment includes a therapeutically effective amount of tDCs generated by the disclosed method for use in treating T1DM. Additionally, a kitembodiment for treating T1DM may comprise a first component containing a therapeutically effective amount of the generated tDCs and a second component containing a therapeutically effective amount of the SCislets or materials for encapsulating SCislets. The kit may further include instructions for administering the therapeutically effective amount of tDCs and therapeutically effective amount of SCislets to enhance the survival and function of the SCislet autografts or allografts.

[0043] In another embodiment, a method for inducing immuno tolerance in a subject with T1DM involves administering a therapeutically effective amount of tDCs generated by the disclosed method and concurrently administering SCislets to promote insulin independence. The administration of tDCs and SCislets may be optimized to mitigate both alloimmune and autoimmune responses in the subject, with the dosage and timing regimen tailored based on the subject's immune profile and the characteristics of the SCislet autografts or allografts.

[0044] Syringable injections to subjects for treatment of T1DM may comprise cells, particles or combinations of both. In some cases, ex vivo manipulated DCs (e.g. tDCs) may be injected. In some cases, ex vivo unmanipulated DCs may be injected. In some cases, a mixture of different types of ex vivo manipulated DCs may be injected such as a mixture of passage 1 and passage 2 cells or a mixture of frozen and fresh cells or a mixture of donor 1 and donor 2 cells or a mixture of subject and donor cells. In some cases, ex vivo manipulated DCs may be injected with other cell populations, such as supporting cell populations.

[0045] In some cases, particles may be injected alone. In some cases, particles containing oligonucleotides (e.g. antisense oligonucleotides) may be injected alone. In some cases, oligonucleotides may be injected alone. In some cases, combinations of cells, oligonucleotides, and particles may be co-injected. In some cases, small molecules, hormones, lipids, proteins such as growth factors, cytokines, chemokines or combinations thereof may be co-injected with cells, oligonucleotides, particles or combinations thereof.

[0046] In the methods, compositions, and kits of this disclosure, tDCs can be delivered by injection. These injections may occur by any route, including intravenous, intramuscular, subcutaneous, intraperitoneal, intrathecal, epidural, intra-arterial, intra-articular, intranodal (e.g. directly into a draining lymph node) and the like.

[0047] Injections may comprise a fluidic phase. In some cases, injections may comprise a solid suspension in a fluidic phase. In some cases, injections may comprise a semi-solid suspension in a fluidic phase. In some cases, injections may comprise a gel suspension in a fluidicphase. In some cases, injections may comprise one or more cells suspended in a fluidic phase. In some cases, injections may comprise one or more particles suspended in a fluidic phase. In some cases, injections may comprise one or more particles and one or more cells suspended in a fluidic phase.

[0048] In some cases, injections may comprise one or more entities (e.g. cells, media, serum, growth factors, cytokines, biomolecules, and the like) in solution in a fluidic phase.

[0049] Subjects may receive about: 0.05x106, 0.1x106, 0.15x106, 0.2x106, 0.25x106, 0.3x106, 0.4x106, 0.5x106, 0.6x 106, 0.7x106, 0.8x106, 0.9x 106, 0.05x107, 0.1x107, 0.15x107, 0.2x107, 0.25x107, 0.3x 107, 0.4x107, 0.5x107, 0.6x107, 0.7x107, 0.8x10, 0.9x107, 0.05x108, 0.1x108, 0.15x108, 0.2x108, 0.25x 108, 0.3x108, 0.4x108, 0.5x 108, 0.6x108, 0.7x108, 0.8x108, or 0.9x108 cells at each injection site. Subjects may receive more than about: 0.05x106, 0.1 x 106, 0.15x106, 0.2x106, 0.25x106, 0.3x106, 0.4x106, 0.5x106, 0.6x106, 0.7x106, 0.8x106, 0.9x106, 0.05x107, 0.1x107, 0.15x107, 0.2x 107, 0.25x 107, 0.3x 107, 0.4x107, 0.5x107, 0.6x107, 0.7x107, 0.8x107, 0.9x107, 0.05x108, 0.1x 108, 0.15x 108, 0.2x108, 0.25x108, 0.3x108, 0.4x108, 0.5x108, 0.6x108, 0.7x108, 0.8x108, 0.9x108 cells at each injection site. Subjects may receive less than about: 0.05x106, 0.1x106, 0.15x106, 0.2x106, 0.25x106, 0.3x106, 0.4x106, 0.5x106, 0.6x106, 0.7x106, 0.8x106, 0.9x106, 0.05x107, 0.1x107, 0.15x107, 0.2x107, 0.25x107, 0.3x107, 0.4x107, 0.5x107, 0.6x107, 0.7x107, 0.8x 107, 0.9x107, 0.05x108, 0.1x108, 0.15x108, 0.2x108, 0.25x108, 0.3x108, 0.4x108, 0.5x108, 0.6x108, 0.7x108, 0.8x108, 0.9x108 cells at each injection site.

[0050] Subjects may receive about: 0.5x107, 0.1x107, 0.15x107, 0.2x107, 0.25x107, 0.3x107, 0.4x107, 0.45x107, 0.5x 107, 0.6x107, 0.75x107, 0.8x 107, 0.9x107, 1.0x107, 1.2x107, or 1.6x107 cells at each of one or more treatments. Subjects may receive more than about: 0.5x107, 0.1x107, 0.15x107, 0.2x107, 0.25x 107, 0.3x 107, 0.4x107, 0.45x107, 0.5x107, 0.6x107, 0.75x107, 0.8x107, 0.9x107, 1.0x107, 1.2x107, or 1.6x107 cells at each of one or more treatments. Subjects may receive less than about: 0.5x107, 0.1x107, 0.15x 107, 0.2x107, 0.25x107, 0.3x107, 0.4x107, 0.45x 107, 0.5x 107, 0.6x 107, 0.75x107, 0.8x107, 0.9x107, 1.0x107, 1.2x107, or 1.6x107 cells at each of one or more treatments.

[0051] Subjects may receive a total cell number between about lx l05-about 6.4x107. Subjects may receive a total cell number of more than between about l xl05-about 6.4x107. Subjects may receive a total cell number of less than between about lx l05-about 6.4x107. In some non-limiting examples, subjects can receive between about 1x106 to about 3X106 cells, orbetween about 1 x 106 to about 5*106 cells, or between about 8*105 to about 4*106 at each of one or more treatments. In some cases, fluorescent imaging results may determine the subsequent number of cellular injections over a treatment course. In some cases, fluorescent imaging results may alter the number of cellular injections over a treatment course. In some cases, fluorescent imaging results may alter the total amount of cells per injection, the frequency of cellular injections over a treatment course, the concentration of cells per injection, or the anatomical location of cellular injections that the subject receives. In some cases, fluorescent imaging results may alter the composition of the injection.

[0052] Subjects may receive about: 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3.0, 3.5, 4.0, 4.5, or 5.0 mg / kg (dry weight) of particles per injection. Subjects may receive more than about: 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3.0, 3.5, 4.0, 4.5, 5.0 mg / kg (dry weight) of particles per injection. Subjects may receive less than about: 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3.0, 3.5, 4.0, 4.5, or 5.0 mg / kg (dry weight) of particles per injection. Subjects may receive about 2 mg / kg (dry weight) of particles per injection.

[0053] Subjects may receive about: 1, 2, 3, 4, 5, 6, 7 particle injections per week. Subjects may receive more than about: 1, 2, 3, 4, 5, 6, 7 particle injections per week. Subjects may receive less than about: 1, 2, 3, 4, 5, 6, 7 particle injections per week. Subjects may receive particles with each cellular injection. In this case, subjects may receive particles in equal frequencies to cellular injection schedules disclosed above. In some cases, fluorescent imaging results may determine the subsequent number of particle injections over a treatment course. In some cases, fluorescent imaging results may alter the number of particle injections over a treatment course. In some cases, fluorescent imaging results may alter the total amount of particles per injection, the frequency of particle injections over a treatment course, the concentration of particles per injection, or the anatomical location of particle injections that the subject receives. In some cases, fluorescent imaging results may alter the composition of the injection.

[0054] The particles can be capable of being injected at a concentration of at least but not limited to about 10 pg of one or more oligonucleotides per mL of the composition being injected. For example, from about 150 to about 500 mg of one or more oligonucleotides may be injectable in a delivery volume of not more than about 1 mL, and generally less than about 2 mL for many applications. The dosage may be divided into two or three or more doses over the day or may be given in a single daily dose.

[0055] In various aspects, the particles may be capable of being injected at a concentration of at least but not limited to about 0.01 to about 1000 mg per mL of the composition being injected. In further aspects, the particles may be capable of being injected at a concentration of at least about: 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 25, 30, 35, 40, 45, or 50 mg per mL or more of the composition being injected. In related aspects, the particles may be capable of being injected at a concentration of at least about: 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335,340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430,435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525,530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620,625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715,720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810,815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905,910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 mg per mL of the composition being injected.

[0056] The volume of a single injection may be about: 0.05, 0.075, 0.1, 0.125, 0.15, 0.175, 0.2, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or 3.5 mL. The volume of a single injection may be more than about: 0.05, 0.075, 0.1, 0.125, 0.15, 0.175, 0.2, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or 3.5 mL. The volume of the injection may be less than about: 0.05, 0.075, 0.1, 0.125, 0.15, 0.175, 0.2, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or 3.5 mL. The volume of the injection may be between about 0.15 and about 0.2 mL. The volume of the injection may be between about 0.5 and about 2 mL.

[0057] In some embodiments, the number of inj ections sites for a single subj ect may be about:1, 2, 3, 4, or 5. In some embodiments, the number of injections sites for a single subject may be more than 1, 2, 3, 4, 5, or more. In some embodiments, the number of injection sites for a single subject may be less than 1, 2, 3, 4, or 5. In some embodiments, the number of injections sites for a single subject may be 4.

[0058] The number of independent delivery treatments may be about: 1, 2, 3, 4, or 5. The number of independent delivery treatments may be more than 1, 2, 3, 4, 5, or more. The numberof independent delivery treatments may be less than 1, 2, 3, 4, or 5. The time between two independent delivery treatments may be about: 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 2 days, 7 days, 14 days, 3 weeks, or 1 month. The time between two independent delivery treatments may be more than 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 2 days, 7 days, 14 days, 3 weeks, or 1 month. The time between two independent delivery treatments may be less than 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 2 days, 7 days, 14 days, 3 weeks, or 1 month. The time between independent delivery treatments can range between 1 and 24 hours. The time between independent delivery treatments can be between 2 days and 1 month. The time between independent delivery treatments can be between 1 week and 3 weeks. The time between independent delivery treatments can be between 1 week and 2 weeks. The time between independent delivery treatments can be between 2 weeks and 3 weeks.

[0059] In the methods, compositions, and kits of this disclosure, one or more injections may be given at any location in the body. One or more injections may be given in the thoracic cavity, abdominal cavity, or the like. One or more injections may be given proximal to the pancreas, for example at a site where lymphatic drainage leads to a pancreatic lymph node, for example preferentially to a pancreatic lymph node. One or more injections may be given proximal to one or more draining lymph nodes. One or more injections may be given proximal to one or more draining lymph nodes located in the abdominal cavity. The term “proximal” may scale with body size. For example, a “proximal” anatomical location in an adult human is at most 5.5 inches from a target site or organ of interest. For example, a “proximal” anatomical location in a pediatric human is at most 2.75 inches from a target site or organ of interest. One or more injections may be on the ventral side of the abdominal cavity. Injections can also be given at a location “superior” to the pancreas. “Superior” refers to the direction towards the head away from the feet. In contrast, “inferior” refers to the direction towards the feet away from the head. Injections can also be given at a location “lateral” from the pancreas (i.e. in the direction away from the midline). The midline of the body runs along the sagittal plane of the body and the pancreas it located on the left side of the body, hence injections given at a location “left and lateral” to the pancreatic lymph node are given on the subject's left side of the body. In rare cases, an individual can have a condition wherein the organs of the body are in a reversed or in mirrored position (i.e. sometimes call organ reversal, situs inversus, situs transversus or oppositus). In individuals with mirrored organs, one skilled in the art will recognize that the right and left terms used herein (e.g. “left and lateral”) would need to be reversed (e.g. “right and lateral), as appropriate for the individual's mirrored organ morphology.

[0060] One or more of the administrations may be subcutaneous or intradermal. One of more of the administrations may be given superior and / or lateral to a pancreatic lymph node.

[0061] In some embodiments, one or more administrations is provided at most about 6 inches superior to the pancreas. In some embodiments, one or more administrations is provided about 4 to about 6 inches superior to a pancreas. In some embodiments, one or more administrations is provided about 4.5 to 5.5 inches superior to a pancreas. In some embodiments, one or more administrations is provided about 5 inches superior to a pancreas. In some embodiments, one or more administrations is provided about: 6, 5.75, 5.5, 5.25, 5, 4.75, 4.5, 4.25, or about 4 inches superior to a pancreas.

[0062] In some embodiments, one or more administrations is provided at most about 3 inches superior to the pancreas. In some embodiments, one or more administrations is provided about 2 to about 3 inches superior to the pancreas. In some embodiments, one or more administrations is provided about 2.25 to 2.75 inches superior to the pancreas. In some embodiments, one or more administrations is provided about 2.5 inches superior to the pancreas. In some embodiments, one or more administrations is provided about: 3, 2.875, 2.75, 2.625, 2.5, 2.375, 2.25, 2.125, or about 2 inches superior to the pancreas.

[0063] In additional embodiments, one or more administrations are provided at most about 4 inches superior to the pancreas. In additional embodiments, one or more administrations are provided at most about 2 inches to about 4 inches superior to the pancreas. In additional embodiments, one or more administrations are provided at most about 2.5 to 3.5 inches superior to the pancreas. In additional embodiments, one or more administrations are provided at most about 3 inches superior to the pancreas. In additional embodiments, one or more administrations are provided at most about: 4, 3.75, 3.5, 3.25, 3, 2.75, 2.5, 2.25, or about 2 inches superior to the pancreas.

[0064] In additional embodiments, one or more administrations are provided at most about 2 inches superior to the pancreas. In additional embodiments, one or more administrations are provided at most about 1 inch to about 2 inches superior to the pancreas. In additional embodiments, one or more administrations are provided at most about 1.25 to 1.75 inches superior to the pancreas. In additional embodiments, one or more administrations are provided at most about 1.5 inches superior to the pancreas. In additional embodiments, one or more administrations are provided at most about: 2, 1.875, 1.75, 1.625, 1.5, 1.375, 1.25, 1.125, or about 1 inch superior to a pancreatic lymph node.

[0065] In further embodiments, one or more administrations are provided about 1.5 to about3.5 inches left and lateral to the pancreas. In further embodiments, one or more administrations are provided about 2 to about 3 inches left and lateral lateral to the pancreas. In further embodiments, one or more administrations are provided about 2.5 inches left and lateral to the pancreas. In further embodiments, one or more administrations are provided about: 1.5, 1.75, 2,2.25, 2.5, 2.75, 3, 3.25, or about 3.5 inches left and lateral to the pancreas.

[0066] In further embodiments, one or more administrations are provided about 0.75 to about 1.75 inches left and lateral to the pancreas. In further embodiments, one or more administrations are provided about 1 to about 1.5 inches left and lateral to the pancreas. In further embodiments, one or more administrations are provided about 1.25 inches left and lateral to the pancreas. In further embodiments, one or more administrations are provided about: 0.75, 0.875, 1, 1.125, 1.25, 1.375, 1.5, 1.625, or about 1.75 inches left and lateral to the pancreas.

[0067] In yet other embodiments, one or more administrations is provided about 4.5 to about6.5 inches left and lateral to the pancreas. In yet other embodiments, one or more administrations is provided about 5 to about 6 inches left and lateral to the pancreas. In yet other embodiments, one or more administrations is provided about 5.5 inches left and lateral to the pancreas. In yet other embodiments, one or more administrations is provided about: 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6,6.25, or about 6.5 inches left and lateral to the pancreas.

[0068] In yet other embodiments, one or more administrations is provided about 2.25 to about 3.25 inches left and lateral to the pancreas. In yet other embodiments, one or more administrations is provided about 2.5 to about 3 inches left and lateral to the pancreas. In yet other embodiments, one or more administrations is provided about 2.75 inches left and lateral to a pancreas. In yet other embodiments, one or more administrations is provided about: 2.25, 2.375, 2.5, 2.625, 2.75, 2.875, 3, 3.125, or about 3.25 inches left and lateral to the pancreas.

[0069] In some instances, the one or more administrations is to a human. In some instances, the subject is a pediatric mammal. In some instances, the subject is not a human (e.g. mouse, nonhuman primate). In some instances, the location of one or more administrations is scaled to body size. In some instances, the location of one or more administrations is scaled to the body size of a patient. In some instances, the location of one or more administrations is scaled to the size of the pancreas of the patient.

[0070] In some embodiments, one or more administrations is provided at most about 6 inches superior to the pancreatic lymph node, such as about 4 to about 6 inches superior to the pancreas,such as about 4.5 to about 5.5 inches superior to the pancreas, such as about 5 inches superior to the pancreas, wherein the one or more administrations is also about 1.5 to about 3.5 inches left and lateral to the pancreas, such as about 2 to about 3 inches left and lateral to the pancreas, such as about 2.5 inches left and lateral to the pancreas.

[0071] In some embodiments, one or more administrations is provided at most about 3 inches superior to the pancreatic lymph node, such as about 2 to about 3 inches superior to the pancreas, such as about 2.25 to about 2.75 inches superior to the pancreas, such as about 2.5 inches superior to the pancreas, wherein the one or more administrations is also about 0.75 to about 1.75 inches left and lateral to the pancreas, such as about 1 to about 1.5 inches left and lateral to the pancreas, such as about 1.25 inches left and lateral to the pancreas.

[0072] In additional embodiments, one or more administrations is provided at most about 6 inches superior to the pancreatic lymph node, such as about 4 to about 6 inches superior to the pancreas, such as about 4.5 to about 5.5 inches superior to the pancreas, such as about 5 inches superior to the pancreas, wherein the one or more administrations is also is provided about 4.5 to about 6.5 inches left and lateral to the pancreas, such as about 5 to about 6 inches left and lateral to the pancreas, such as about 5.5 inches left and lateral to the pancreas.

[0073] In additional embodiments, one or more administrations is provided at most about 3 inches superior to the pancreatic lymph node, such as about 2 to about 3 inches superior to the pancreas, such as about 2.25 to about 2.75 inches superior to a pancreatic lymph node, such as about 2.5 inches superior to a pancreatic lymph node, wherein the one or more administrations is also is provided about 2.25 to about 3.25 inches left and lateral to the pancreas, such as about 2.5 to about 3 inches left and lateral to the pancreas, such as about 2.75 inches left and lateral to the pancreas.

[0074] In other embodiments, one or more administrations is provided at most about 4 inches superior to the pancreas, such as about 2 inches to about 4 inches superior to the pancreas, such as about 2.5 to about 3.5 inches superior to the pancreas, such as about 3 inches superior to the pancreas, wherein the one or more administrations is about 4.5 to about 6.5 inches left and lateral to the pancreas, such as about 5 to about 6 inches left and lateral to the pancreas, such as about 5.5 inches left and lateral to a the pancreas.

[0075] In other embodiments, one or more administrations is provided at most about 2 inches superior to the pancreas, such as about 1 inch to about 2 inches superior to the pancreatic lymph node, such as about 1.25 to about 1.75 inches superior to the pancreas, such as about 1.5 inchessuperior to the pancreas, wherein the one or more administrations is about 2.25 to about 3.25 inches left and lateral to the pancreas, such as about 2.5 to about 3 inches left and lateral to the pancreas, such as about 2.75 inches left and lateral to the pancreas.

[0076] In other embodiments, one or more administrations is provided at most about 4 inches superior to the pancreas, such as about 2 inches to about 4 inches superior to the pancreas, such as about 2.5 to about 3.5 inches superior to the pancreas, such as about 3 inches superior to the pancreas, wherein the one or more administrations is also about 1.5 to about 3.5 inches left and lateral to the pancreas, such as about 2 to about 3 inches left and lateral to the pancreas, such as about 2.5 inches left and lateral to the pancreas.

[0077] In other embodiments, one or more administrations is provided at most about 2 inches superior to the pancreas, such as about 1 inch to about 2 inches superior to the pancreatic lymph node, such as about 1.25 to about 1.75 inches superior to the pancreas, such as about 1.5 inches superior to the pancreas, wherein the one or more administrations is also about 0.75 to about 1.75 inches left and lateral to the pancreas, such as about 1 to about 1.5 inches left and lateral to the pancreas, such as about 1.25 inches left and lateral to the pancreas.

[0078] Administrations can be used in combination. In some non-limiting examples, 1, 2, 3 or 4 administrations are provided to the subject, wherein each administration is in a different location from the other administration, in reference to the pancreas. In some non-limiting examples, the injection is subcutaneous. In additional specific non-limiting examples, the injection is subcutaneous. A diagram, showing one exemplary non-limiting administration protocol is provided in FIG. 19. In some instances, the one or more administrations is to a human. In some instances, the one or more administrations is to a pediatric patient. In some instances, the subject is not a human (e.g. mouse, non-human primate). In some instances, the location of one or more administrations is scaled to body size.

[0079] One or more injections may be given about 5.5 inches left and lateral to the pancreatic lymph node and about 3 inches superior to the pancreatic lymph node. One or more injections may be given about 2.5 inches left and lateral to the pancreatic lymph node and about 3 inches superior to the pancreatic lymph node. One or more injections may be given about 5.5 inches left and lateral to the pancreatic lymph node and about 5 inches superior to the pancreatic lymph node. One or more injections may be given about 2.5 inches left and lateral to the pancreatic lymph node and about 5 inches superior to the pancreatic lymph node.

[0080] One or more injections may be given about 2.75 inches left and lateral to the pancreatic lymph node and about 1.5 inches superior to the pancreatic lymph node. One or more injections may be given about 1.25 inches left and lateral to the pancreatic lymph node and about 1.5 inches superior to the pancreatic lymph node. One or more injections may be given about 2.75 inches left and lateral to the pancreatic lymph node and about 2.5 inches superior to the pancreatic lymph node. One or more injections may be given about 1.25 inches left and lateral to the pancreatic lymph node and about 2.5 inches superior to the pancreatic lymph node.

[0081] The location of the pancreatic lymph nodes can readily be determined by one of skill in the art. In specific non-limiting examples, imaging is used, such as computed tomography (CT) is used. In additional non-limiting examples, multidetector computed tomography (MDCT), endoscopic ultrasound (EUS), magnetic resonance imaging (MRI), radionucleotide imaging, [18F]-fluorodeoxyglucose positron emission tomography (FDG-PET) scanning, Optical coherence tomography (OCT) can be used to determine the location of the pancreas and the pancreas. However, imaging need not be used, and an expected anatomic location of the pancreatic lymph node can be determined by anatomic landmarks on the abdomen that indicate a location where the pancreatic lymph node is typically located. In some examples, the injections are to the left of the midline, and superior to the umbilicus.

[0082] Imaging, such as computed tomography (CT) may be used to collect imaging data on the location of oligonucleotides, particles, cells, or combinations thereof injected into the body. In type 1 diabetes applications, imaging may be used to collect imaging data on whether injected oligonucleotides, particles, cells, or combinations thereof remain near the pancreas or migrate to other organs such as mesenteric lymph nodes, spleen, large intestine, liver, adipose tissue, thymus, lung, kidney, or others. Imaging may be used to collect imaging data on whether injected oligonucleotides, particles, cells or combinations thereof remain near the tissue of interest, such as the pancreas. Imaging may be used to collect imaging data on whether injected cells remain viable after injection. Imaging may be used to collect imaging data on the location of oligonucleotides, particles, cells or combinations thereof at specific times after injection. Imaging data may be used to determine the timing of subsequent injections. Imaging data may be used to determine the location of subsequent injections. Imaging data may be used to determine the composition of subsequent injections. Imaging data may be used to determine the amount of subsequent injections. Imaging data may be used to determine the efficacy of the current injection. Imaging data may be used to determine the percentage of oligonucleotides, particles, cells, or combinations thereof that accumulate in specific organs such as the pancreas. Imaging data maybe used to determine the rate of accumulation or the rate of dispersion of oligonucleotides, particles, cells or combinations thereof in a given tissue.

[0083] Imaging may be collected continuously in real-time. Imaging may be one or more discrete images taken at specific times. Additional non-limiting imaging examples include, multidetector computed tomography (MDCT), endoscopic ultrasound (EUS), magnetic resonance imaging (MRI), radionucleotide imaging. [18F]-fluorodeoxyglucose positron emission tomography (FDG-PET) scanning, or Optical coherence tomography (OCT).

[0084] One or more particles may be labeled with one or more moieties to enable image tracking. One or more particles may be labeled with a color tracking marker, a radio-active tracking marker, a pH indicator, or combinations thereof. One or more particles may be labeled with one or more fluorescent moieties. One or more particles may be labeled with a radio-active moiety. All particles may be labeled. A subset of particles may be labeled. Subsets of particles may be labeled differently. Particles may be labeled to enable recovery and analysis following injection.

[0085] One or more cells may be labeled with one or more moieties to enable image tracking. One or more cells may be labeled with a color tracking marker, a radio-active tracking marker, a viability marker, a surface marker, an antigen, or combinations thereof. One or more cells may be labeled with one or more fluorescent moieties. One or more cells may be labeled with one or more radio-active moieties. All cells may be labeled. A subset of cells may be labeled. Subsets of cells may be labeled differently. Cells may become labeled from uptake of labeled particles. Cells may be labeled to enable recovery and analysis following injection.

[0086] One or more oligonucleotides may be labeled with one or more moieties to enable image tracking. One or more oligonucleotides may be labeled with a color tracking marker, a radio-active tracking marker, or combinations thereof. One or more oligonucleotides may be labeled with one or more fluorescent moieties. One or more oligonucleotides may be labeled with one or more radio-active moieties. All oligonucleotides may be labeled. A subset of oligonucleotides may be labeled. Subsets of oligonucleotides may be labeled differently.

[0087] A suitable delivery route may be injection with a fine bore needle, which includes subcutaneous, ocular and the like. The term “fine bore needle” may mean needles of at least 20 gauge size, typically between about 22 gauge and about 30 gauge and above. In some cases, the fine bore needle may be least as fine as 24 gauge, at least as fine bore as 26 gauge, and at least as fine as 28 gauge.

[0088] Injection delivery is made during a normal injection time period. In some cases, such time periods may be about: 5, 10, 15, 20, or 25 seconds. In some cases, such time periods may be may be less than about: 5, 10, 15, 20, 25 seconds or less. In some cases, such time periods may be more than about: 5, 10, 15, 20, 25 seconds or more.

[0089] The two or more subcutaneous injections administered at one or more injection sites proximal to the pancreas, may bring blood glucose levels closer to a pre-diabetic level for about:1 day, 7 days, 21 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, or 20 months. In some embodiments, blood glucose levels may be brought closer to a pre-diabetic levels for at least about: 1 day, 7 days, 21 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months,10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months or more. In some embodiments, blood glucose levels may be brought closer to a pre-diabetic levels for at most about: 1 day, 7 days, 21 days, 30 days, 1 month,2 months, 3 months, or 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months,11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, or 20 months. The two or more subcutaneous injections administered at one or more injection sites proximal to a pancreatic lymph node or the pancreas, may bring blood glucose levels closer to a pre-diabetic level for between about 25 to about 35 days. The two or more subcutaneous injections administered at one or more injection sites proximal to a pancreatic lymph node or the pancreas, may bring blood glucose levels closer to a pre-diabetic level for between about 28 to about 32 days. The two or more subcutaneous injections administered at one or more injection sites proximal to a pancreatic lymph node or the pancreas, may bring blood glucose levels closer to a pre-diabetic level for between about 20 to about 40 days. The two or more subcutaneous injections administered at one or more injection sites proximal to a pancreatic lymph node or the pancreas, may bring blood glucose levels closer to a pre-diabetic level for between about 65 to about 75 weeks. The two or more subcutaneous injections administered at one or more injection sites proximal to a pancreatic lymph node or the pancreas, may bring blood glucose levels closer to a pre-diabetic level for between about 68 to about 72 weeks. The two or more subcutaneous injections administered at one or more injection sites proximal to a pancreatic lymph node or the pancreas, may bring blood glucose levels closer to a pre-diabetic level for between about 60 to about 80 weeks. Four subcutaneous injections administered at 4 injection sites proximal to the pancreatic lymph node, may bring blood glucose levels closer to pre-diabeticlevels for about 70 weeks or more. Bringing blood glucose levels closer to pre-diabetic levels can be restoring blood glucose levels to pre-diabetic levels.

[0090] The two or more subcutaneous injections administered at one or more injection sites proximal to a pancreatic lymph node or the pancreas, may preserve remaining pancreatic beta cell viability for about: 7 days, 21 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, or 20 months. In some embodiments, remaining pancreatic beta cells may be preserved for at least about: 7 days, 21 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months or more. In some embodiments, remaining pancreatic beta cells may be preserved for at most about: 7 days, 21 days, 30 days, 1 month, 2 months, 3 months, or 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, or 20 months. The two or more subcutaneous injections administered at one or more injection sites proximal to a pancreatic lymph node or the pancreas, may preserve remaining pancreatic beta cell viability for between about 25 to about 35 days. The two or more subcutaneous injections administered at one or more injection sites proximal to a pancreatic lymph node or the pancreas, may preserve remaining pancreatic beta cell viability for between about 65 to about 75 weeks. Four subcutaneous injections administered at 4 injection sites proximal to the pancreatic lymph node, may preserve remaining pancreatic beta cell viability for about 70 weeks or more.

[0091] These embodiments are exemplary and are not intended to be limiting. Variations and modifications of these embodiments may be made without departing from the spirit and scope of the disclosure. Various of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art, each of which is also intended to be encompassed by the disclosed embodiments.EXAMPLES

[0092] Example 1 - Islet allograft dendritic cell immunoprotection

[0093] Cadaveric islet allotransplantation is widely performed in many countries and is FDA- approved in the United States as a biologic treatment to reverse type 1 diabetes and thereby obviate the need for further insulin replacement therapy (Witkowski 2025). However lifelong systemicimmunosuppression is required (Spence 2025) to protect the allografted islets against both the underlying islet P-cell autoimmunity of type 1 diabetes and secondary allo-immunity associated with cadaveric organ transplantation (Czarnecka 2023). This requirement exposes the recipient to greatly heightened risk of serious and potentially life-threatening infection and immunosuppressive drugs paradoxically may be in themselves toxic to the allotransplanted islets, so that pharmacologic systemic immunosuppression is considered one of the greatest barriers to islet cell replacement therapy (Czarnecka 2023). Published clinical (Giannoukakis 2011) and preclinical (Machen 2004) evidence would suggest that recipient-derived DC’s artificially rendered tolerogenic by antisense deoxyribonucleotide (AS-ODN) mediated suppression of CD40, CD80 and CD86 surface protein expression theoretically could protect cadaveric islet allografts against the allogeneic immune response as well as the intrinsic islet P-cell autoimmunity exhibited by type 1 diabetic patients; (Figure 1) would suggest that these tolerogenic dendritic cells can also protect islet allografts against allo-immunity. These data demonstrate that while AS- ODN DCs are only marginally superior to standard GM-CSF+IL-4- generated immature "control" DCs in allograft protection, they are rendered clearly superior to these control tolerogenic DCs once exposed to and immunologically informed by the allograft lysate (Figure 1). Figure 1 depicts the effect of AS-ODN modified DC’s and exposure to allograft lysate on preserving islet cell allograft function and normoglycemia in streptozotocin diabetic mice. Five days prior to transplant, Balb / c recipient mice were pretreated with 106lysate-exposed or lysate-unexposed Balb / c DC's or saline injected intraperitoneally, and two days thereafter rendered diabetic with streptozotocin. Three days thereafter, the Balb / c recent mice received 350 islets isolated from donor C3H / HeJ mice under the kidney capsule. The recipient mice were retreated intraperitoneally with 106lysate-exposed or unexposed DC's or saline 2-, 4-, 6- & 8-weeks post-transplants. Blood glucose was measured periodically to determine the percentage of mice in each treatment group that remained normoglycemic post islet cell allograft placement. For DC lysate exposure, 500 freshly collected islets were washed in PBS and then underwent 5 cycles of freeze-thaw (freeze in liquid nitrogen followed by thaw in a 37 Celsius water bath. The lysate was then centrifuged at 2000 x g for 10 minutes at 4 degrees to clarify. The clarified lysate was then passed through a 0.2-micron filter and protein concentrations was measured by microassay. Clarified lysate was separated into aliquots which were then frozen at -80 Celsius. On the day of DC pulsing, the islet lysate aliquot was thawed to room temperature. 100 micrograms were added to the DC cultures (day 5 cultures; 1-2 x 106cells per flask) for a 24-hour incubation. The DC’s were then washed three times in PBS and resuspended in PBS at a final number of 1-2 x 106cells in a minimal injection volume. Taken together, these published and unpublished preclinical and clinical observations strongly suggest that these AS-ODN-generated tDC’s might enable successful clinical islet allograft transplantation and preserved graft function in patientswith type 1 diabetes while obviating or greatly reducing the need for chronic systemic pharmacologic immunosuppression. However, the translation of these results into clinical application would require further investigation. The specific protocol(s) by which tDC immunoprotection would be integrated into cadaveric islet cell allograft implantation protocols (Czamecka 2023, Lantidra 2023, Spence 2025) remain to be established, and would require preliminary preclinical experimentation before translation to the clinic. Current protocols for human cadaveric islet transplantation involve identification of a ABO-blood-group and human leukocyte antigen compatible T- and B-lymphocyte cross-matched (Lantidra 2023) non-diabetic deceased pancreas donor, pancreatectomy and tissue digestion, islet isolation and culture for up to 72 hours, and implantation into the liver by infusion of the cultured islets into the portal vein of the recipient (Rickels 2019). Following donor pancreatectomy, pancreas digestion and islet isolation, islets generally should be cultured for less than 72 hours, whereupon the resulting infusion-ready islet suspension should be infused into the recipient within 6 hours (Rickels 2019, Lantidra 2023). This would in general preclude pre-transplantation treatment of the recipient with allograft lysate- exposed AS-ODN-induced tolerogenic dendritic cells 5 days prior to transplantation as was done in preliminary mouse experiments (Figure 1). Thus, it is likely that some elements of current acute immunosuppressive therapy of islet allograft recipients (Figure 2) would be retained in conjunction with long-term AS-ODN lysate-exposed dendritic cell treatment that would obviate the need for long-term chronic immunosuppression. Figure 2 depicts peritransplant management for islet allotransplantation. At the time, compatible islet preparation is available and enters culture, induction and maintenance immunosuppression is initiated in the hospital where intensive insulin therapy is maintained targeting normoglycemia. At the time of islet infusion, antiinflammatory therapy (e.g., etanercept) and anticoagulation are initiated, with unfractionated heparin transitioned to a subcutaneous regimen with antiplatelet therapy using low-dose aspirin added by the second day. (Shown is the CIT07 protocol from Hering BJ, Clarke WR, Bridges ND, et al. Phase 3 trial of transplantation of human islets in type 1 diabetes complicated by severe hypoglycemia. Diabetes Care 2016;39:1230-1240; other approaches may vary.) From Rickels 2019. Furthermore, human islet transplantation is performed by infusion into the portal vein and islet lodgment within the liver, in contradistinction to the renal subcapsular injection utilized in rodents (portal infusion / injection is considered exceedingly difficult mice). Therefore, the location as well as the timing of tolerogenic dendritic cells would need to be explored further in larger preclinical species. Thus, various aspects of these management protocols need to be explored and optimized in preclinical animal models of islet allotransplantation prior to the introduction of this approach into the clinic.

[0094] Bibliography

[0095] Czarnecka 2023. Czarnecka, Z.; Dadheech, N.; Razavy, H.; Pawlick, R.; Shapiro, A.M.J. The Current Status of Allogenic Islet Cell Transplantation. Cells 2023, 12, 2423. https: / / doi.org / 10.3390 / cellsl2202423.

[0096] Giannoukakis 2011. Giannoukakis N et al. Phase I (Safety) Study of Autologous Tolerogenic Dendritic Cells in Type 1 Diabetic Patients. Diabetes Care 34:2026-2032, 2011.

[0097] Lantidra 2023. LANTIDRA (donislecel-jujn) Allogeneic Pancreatic Islet Cellular Suspension for hepatic portal vein infusion. Initial US Approval: 2023. CellTrans Inc. 1740 W. Taylor St., STE C200 Chicago, IL, 60612.

[0098] Li 2014. Li Y, Liu M, Yang S-T. Dendritic cells derived from pluripotent stem cells: Potential of large scale production. World J Stem Cells 2014 January 26; 6(1): 1-10.

[0099] Machen 2004. Machen J, Harnaha J, Lakomy R, Styche A, Trucco M, Giannoukakis N. Antisense oligonucleotides down-regulating costimulation confer diabetes-preventive properties to nonobese diabetic mouse dendritic cells. J Immunol 2004;173:4331-4341.

[0100] Rickels 2019. Rickets MR, Robertson RR Pancreatic Islet Transplantation in Humans: Recent Progress and Future Directions. Endocrine Reviews 40: 631 - 668, 2019.

[0101] Spence 2025. Spence KT Ladie DE. Islets Transplantation. StatPearls [Internet], Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Bookshelf ID: NBK562272 PMID: 32965943

[0102] Witkowski 2025. Witkowski P, Wojcik N, Appelbaum N, FungJJ, Barth RN, and Ricordi C (2025). Demise of cadaveric islet transplantation in the USA: Quo Vadis, 1 year after BLA approval and 24 years after the Edmonton breakthrough? Front. Transplant. 4: 1491568. doi: 10.3389 / frtra.2025.1491568.

[0103] Various implementations have been described, and it will be understood that modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other implementations are within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method for treating Type 1 diabetes mellitus (T1DM) comprising: generating tolerogenic dendritic cells (tDCs) by downregulating expression of genes involved in an adaptive immune response in dendritic cells (DCs); exposing said tDCs to antigens associated with stem cell-derived P-cells (SCislets) or to materials used for encapsulating SCislets to induce autografts or allograft-specific immunotolerance; and administering said tDCs to a subject in conjunction with placement of SCislet autografts or allografts to enhance survival and function of the SCislet autografts or allografts.

2. The method of claim 1, wherein the genes involved in the adaptive immune response include CD40, CD80, and CD86.

3. The method of claim 1, wherein the tDCs are generated by exposure to antisense oligonucleotides, short interfering ribonucleic acids (siRNAs), or through gene editing technologies.

4. The method of claim 3, wherein the gene editing technology is CRISPR / Cas9.

5. The method of claim 1, wherein the tDCs are derived from a recipient of the SCislet autografts or allografts.

6. The method of claim 1, wherein the tDCs are derived from the same induced pluripotent human stem cell (ihPSC) source as the SCislets.

7. The method of claim 1, wherein the tDCs are administered systemically.

8. The method of claim 1, wherein the tDCs are administered locally at or around the SCislet autografts or allograft site.

9. The method of claim 1, wherein the tDCs are administered before, during, or after the placement of the SCislet autografts or allografts.

10. The method of claim 1, further comprising encapsulating the SCislet autografts or allografts prior to placement.

11. The method of claim 10, wherein the encapsulating material is selected to promote host vascular invasion and vascularization of the SCislet autografts or allografts.

12. The method of claim 1, further comprising a step of pre-exposing the tDCs to stress-induced antigens expressed by the SCislets during post-implantation maturation.

13. The method of claim 1, wherein the tDCs are administered in a formulation designed to target dendritic cells.

14. The method of claim 1, wherein the tDCs are administered in conjunction with microspheres containing oligonucleotides for in vivo generation of immuno tolerant dendritic cells.

15. The method of claim 1, further comprising administering an immunosuppressant.

16. The method of claim 15, wherein the immunosuppressant is selected from tacrolimus, sirolimus, etanercept, thymoglobulin or any combination thereof.

17. The method of claim 15, wherein the immunosuppressant is administered before, during, or after the administration of the tDCs and SCislet autografts or allografts.

18. The method of claim 1, further comprising administering insulin therapy.

19. The method of claim 18, wherein the insulin therapy is administered before, during, or after the administration of the tDCs and SCislet autografts or allografts.

20. The method of claim 1, further comprising administering an anticoagulant.

21. The method of claim 20, wherein the anticoagulant is heparin.

22. The method of claim 20, wherein the anticoagulant is administered before, during, or after the administration of the tDCs and SCislet autografts or allografts.

23. The method of claim 1, further comprising administering aspirin.

24. The method of claim 20, wherein the aspirin is administered before, during, or after the administration of the tDCs and SCislet autografts or allografts.

25. A pharmaceutical composition comprising tolerogenic dendritic cells (tDCs) generated by the method of claim 1 for use in treating Type 1 diabetes mellitus (T1DM).

26. A kit for treating Type 1 diabetes mellitus (T1DM) comprising: a first component containing tolerogenic dendritic cells (tDCs) generated by the method of claim 1; and a second component containing stem cell-derived P-cells (SCislets) or materials for encapsulating SCislets.

27. The kit of claim 26, further comprising instructions for administering the tDCs and SCislets to a subject in a manner that enhances the survival and function of the SCislet autografts or allografts.

28. A method for inducing immunotolerance in a subject with Type 1 diabetes mellitus (T1DM) comprising: administering a therapeutically effective amount of tolerogenic dendritic cells (tDCs) generated by the method of claim 1 ; and concurrently administering stem cell-derived [l-cclls (SCislets) to the subject to promote insulin independence.

29. The method of claim 28, wherein the tDCs and SCislets are administered in a manner that mitigates both alloimmune and autoimmune responses in the subject.

30. The method of claim 28, wherein the tDCs are administered in a dosage and timing regimen optimized based on the subject's immune profile and the characteristics of the SCislet autografts or allografts.

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