Yan Li

Stem cell therapy holds significant potential for many inflammatory diseases and regenerative medicine applications. However, delivery of therapeutic cells to specific disease sites after systemic administration without indiscriminate trafficking to other non-target tissues is a major limitation of current cell therapies. Here, we describe a novel nanocarrier-directed targeted cell delivery system that enables cell surface coating with dendrimer nanocarriers containing adhesion moieties to serve as a global positioning system “GPS” to guide circulating cells to targeted lesions and mediate the anchoring of cells at the inflammation site. By exploiting cell surface ligands/receptors selectively and/or molecular moieties that are highly expressed on activated endothelium in pathologic disease states, nanocarrier-coated cells containing the counterpart binding receptors/ligands can be enabled to specifically traffic to and dock at vasculature within target lesions. We demonstrate the efficacy of the I-domain fragment of LFA-1 (idLFA-1) complexed to modified nanocarriers to facilitate the homing of mesenchymal stem cells (MSCs) to inflamed luminal endothelial cells on which ICAM-1 is highly expressed in a murine model of aortic atherosclerosis. Our method can overcome challenges imposed by the high velocity and dynamic circulatory flow of the aorta to successfully deliver MSCs to atherosclerotic regions and allow for the docking of the potentially therapeutic and immunomodulating cells. This targeted cell delivery platform can be tailored for selective systemic delivery of various types of therapeutic cells to different disease areas.

FANCA is one of the 23 genes whose deficiencies lead to defective DNA interstrand crosslink repair and cancer-prone Fanconi anemia disease. Beyond its functions in DNA repair and tumor suppression, we report that high FANCA expression is strongly associated with breast cancer development. Overexpression of WT-FANCA significantly promotes breast cancer cell proliferation and tumor growth both in vitro and in vivo, while FANCA deficiency severely compromises the proliferation of breast cancer cells, but not non-tumorigenic breast epithelial cells. Heterozygous knockout of FANCA in breast cancer mouse models is sufficient to cause significant reduction of breast tumor growth in vivo. Furthermore, we have shown that high FANCA expression in breast cancer correlates with promoter hypomethylation in a TET-dependent manner, and TET inhibition recapitulates the proliferation defects caused by FANCA deficiency. Our study identifies the oncogenic properties of WT-FANCA and shows that FANCA is a promising target for breast cancer intervention.
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•High expression of WT-FANCA promotes breast cancer cell and tumor growth in vitro and in vivo•FANCA deficiency specifically disrupts breast cancer cell-cycle progression and tumor growth•Hypomethylation of the FANCA promoter accounts for its high expression in breast cancer
FANCA is a DNA repair gene that plays a tumor-suppressor role by maintaining genome stability in normal cells. Luo et al. report that instead of being a tumor suppressor, WT-FANCA demonstrates oncogenic properties in breast cancer by promoting cell proliferation, and it is targetable for breast cancer intervention.

Objective: This study focuses on dose-response investigation using a codon-optimized and de novo -synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug (IND)-enabling of subsequent clinical studies. Background: Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia (CLTI). Understanding the dose for effective gene delivery is crucial for future IND-enabling studies. Methods: Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved three cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly (IM) in divided aliquots, ranging from 2×10 9 VG to 2×10 11 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery and therapeutic angiogenesis were assessed. Results: Codon-optimized E-Sel/AAV2 gene therapy exhibits superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo . Mice treated with a high dose (2×10 11 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber’s scores, increased running stamina and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied. Conclusion: E-Sel/AAV2 Vascular Regeneration Gene Therapy (VRGT) holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2×10 11 VG aliquots injected IM.

Abstract only Introduction: It is estimated that about 40 to 60 million people in the United States suffer from diabetic foot ulcers (DFUs), one of the most severe DM complications. A significant proportion of affected individuals undergo amputation and are exposed to high mortality risks. Stem cell-based therapies offer a promising therapeutic avenue for these patients. Fibroblasts hold a crucial role in wound healing and angiogenesis, and we further highlighted that activated Notch signaling in fibroblasts derived from diabetic wounds leads to dysregulated fibroblast function. In this study, the potential therapeutic outcomes from genetically modified fibroblasts derived from mesenchymal stem cells (MSC-DF) in treating DFU is assessed, utilizing a unique genetic mouse model. Methods: A fibroblast-specific and inducible diabetic murine model with triple-gene engineering was created via inducible intracellular Notch1 pathway activation (RosaN1IC/Col1a2-Cre/ERT/Leprdb). MSC-DFs harvested from 10-12 week old B6db/ ROSAN1I/ / Cola2-Cre/ERT (LLRRCc) donor mice were cultured in vitro with activation versus silencing of Notch 1 signaling. Recipient LLRRCc mice (8-12-week-old) underwent a 6mm excisional dorsal skin wound and subsequent local injection of 1x10^6 donor syngeneic activated or silenced GOF Notch1 -MSC-DFs (n= 5/group). Wound closure was monitored daily for 9 consecutive days post-injury, and tissue was obtained for histological sectioning. Results: GOF Notch1 -MSC-DFs were characterized by immunostaining based on expression of markers specific to fibroblasts and a panel of Notch signaling genes. LLRRCc murine subjects receiving silenced GOF Notch1 -MSC-DFs treatment (n=5) demonstrated faster rates of wound closure over activated GOF Notch1 -MSC-DFs (n=5) treated mice. Collagen deposition was more abundant in wounds treated with silenced GOF Notch1 -MSC-DFs. Conclusion: MSC-DF deactivated Notch1 signaling therapy improves the rate of wound healing in GOF Notch Diabetic mice. These promising outcomes demonstrate a potential role for this innovative stem cell-based therapy targeting Notch-1-modified MSC-DF in future clinical applications for DFU patients through restoration of impaired fibroblast function.

Stem cell therapy holds significant potential for many inflammatory diseases and regenerative medicine applications. However, delivery of therapeutic cells to specific disease sites after systemic administration without indiscriminate trafficking to other non-target tissues is a major limitation of current cell therapies. Here, we describe a novel nanocarrier-directed targeted cell delivery system that enables cell surface coating with dendrimer nanocarriers containing adhesion moieties to serve as a global positioning system "GPS" to guide circulating cells to targeted lesions and mediate the anchoring of cells at the inflammation site. By exploiting cell surface ligands/receptors selectively and/or molecular moieties that are highly expressed on activated endothelium in pathologic disease states, nanocarrier-coated cells containing the counterpart binding receptors/ligands can be enabled to specifically traffic to and dock at vasculature within target lesions. We demonstrate the efficacy of the I-domain fragment of LFA-1 ( LFA-1) complexed to modified nanocarriers to facilitate homing of mesenchymal stem cells (MSCs) to inflamed luminal endothelial cells on which ICAM-1 is highly expressed in a murine model of aortic atherosclerosis. Our method can overcome challenges imposed by the high velocity and dynamic circulatory flow of the aorta to successfully deliver MSCs to atherosclerotic regions and allow for docking of the potentially therapeutic and immunomodulating cells. This targeted cell-delivery platform can be tailored for selective systemic delivery of various types of therapeutic cells to different disease areas.

OBJECTIVEHere we report a new method to increase therapeutic potential of mesenchymal stem/stromal cells (MSCs) for ischemic wound healing. We tested biologic effects of MSCs modified with E-selectin, a cell-adhesion molecule capable of inducing post-natal neovascularization, on a translational murine model. SUMMARY BACKGROUND DATATissue loss significantly worsens the risk of extremity amputation for patients with chronic limb-threatening ischemia (CLTI). MSCs-based therapeutics hold major promise for wound healing and therapeutic angiogenesis, but unmodified MSCs demonstrate only modest benefits. METHODSBone marrow cells harvested from FVB/ROSA26SormTmG donor mice were transduced with E-selectin-GFP/AAV-DJ or GFP/AAV-DJ (control). Ischemic wounds were created via a 4 mm punch biopsy in the ipsilateral limb after femoral artery ligation in recipient FVB mice and subsequently injected with PBS or 1×10^6 donor MSCGFP or MSCE-selectin-GFP. Wound closure was monitored daily for 7 postoperative days (POD), and tissues were harvested for molecular and histological analysis and immunofluorescence. Whole-body DiI perfusion and confocal microscopy was utilized to evaluate wound angiogenesis. RESULTSUnmodified MSCs do not express E-selectin, and MSCE-selectin-GFP gain stronger MSC phenotype yet maintain trilineage differentiation and colony-forming capability. MSCE-selectin-GFP therapy accelerates wound healing compared to MSCGFP and PBS treatment. Engrafted MSCE-selectin-GFP manifest stronger survival and viability in wounds at POD 7. Ischemic wounds treated with MSCE-selectin-GFP exhibit more abundant collagen deposition, and enhanced angiogenic response. CONCLUSIONWe establish a novel method to potentiate regenerative and pro-angiogenic capability of MSCs by modification with E-selectin/AAV. This innovative therapy carries potential as a platform worthy of future clinical studies.

The response to ischemia in peripheral artery disease (PAD) depends on compensatory neovascularization and coordination of tissue regeneration. Identifying novel mechanisms regulating these processes is critical to the development of nonsurgical treatments for PAD. E-selectin is an adhesion molecule that mediates cell recruitment during neovascularization. Therapeutic priming of ischemic limb tissues with intramuscular E-selectin gene therapy promotes angiogenesis and reduces tissue loss in a murine hindlimb gangrene model. In this study, we evaluated the effects of E-selectin gene therapy on skeletal muscle recovery, specifically focusing on exercise performance and myofiber regeneration. C57BL/6J mice were treated with intramuscular E-selectin/adeno-associated virus serotype 2/2 gene therapy (E-sel/AAV) or LacZ/AAV2/2 (LacZ/AAV) as control and then subjected to femoral artery coagulation. Recovery of hindlimb perfusion was assessed by laser Doppler perfusion imaging and muscle function by treadmill exhaustion and grip strength testing. After three postoperative weeks, hindlimb muscle was harvested for immunofluorescence analysis. At all postoperative time points, mice treated with E-sel/AAV had improved hindlimb perfusion and exercise capacity. E-sel/AAV gene therapy also increased the coexpression of MyoD and Ki-67 in skeletal muscle progenitors and the proportion of Myh7+ myofibers. Altogether, our findings demonstrate that in addition to improving reperfusion, intramuscular E-sel/AAV gene therapy enhances the regeneration of ischemic skeletal muscle with a corresponding benefit on exercise performance. These results suggest a potential role for E-sel/AAV gene therapy as a nonsurgical adjunct in patients with life-limiting PAD.

Abstract only Objective: Tissue loss considerably worsens the risk of extremity amputation in patients with chronic limb-threatening ischemia (CLTI), and unmodified mesenchymal stem cell (MSC) therapies have failed to demonstrate more than minor clinical benefits for inducing therapeutic angiogenesis and wound healing. Here we describe the effects of MSCs engineered to overexpress E-selectin, a cell-adhesion molecule capable of stimulating neovascularization, using a translational murine model recapitulating hindlimb ischemia with cutaneous tissue loss. Methods: MSCs were harvested from bone marrow of 8-10-week-old FVB/Rosa26Sor mTmG donor mice and verified as CD44 + /CD73+/CD105 + /CD29 + /Sca-1 + by flow cytometry. MSCs underwent viral transduction with E-selectin-GFP/AAV or GFP/AAV. Femoral artery ligation was performed in 12-14-week-old recipient FVB mice followed by a 4 mm cutaneous wound in the ipsilateral limb and local injection of PBS or 1x10^6 donor GFP + /MSCs or E-selectin + /MSCs (E-sel + /MSC). Wounds were monitored daily for 7 days and then harvested. Results: Unmodified MSCs do not express E-selectin, and E-sel + /MSCs retained MSC phenotype. Mice receiving E-sel + /MSC treatment (n=10) demonstrated accelerated wound closure compared to both GFP + /MSCs (n=10) and PBS (n=10) treated mice at each post-operative day (POD) with the highest degree of difference observed at POD 5 (94 ± 3% vs. 79 ± 10% GFP vs. 72 ± 9% PBS, p<0.001) and 7 (98 ± 2% vs. 86 ± 8% GFP vs. 87 ± 6% PBS; p<0.010). Collagen deposition was greater in wounds treated with E-sel + /MSCs (21 ± 4% vs. 5 ± 1% GFP vs. 9 ± 1% PBS). DiI perfusion at POD 10 demonstrated greater vessel density in wounds treated with E-sel + /MSCs (76 ± 15% vs. 29 ± 7% GFP vs. 9 ± 5% PBS relative vascular density). E-sel + /MSCs demonstrated improved viability with more mTmG + -E-sel + /MSCs (13 ± 3 cells/high powered field (HPF) vs. GFP + /MSCs 3 ± 2 cells/HPF vs. PBS 0 ± 0 cells/HPF, p<0.010) at POD 7 on immunofluorescent microscopy. Conclusion: Cell therapy using E-selectin-overexpressing MSCs overcomes impairments in wound healing of ischemic limbs. These data exhibit the potential role for E-selectin-modified MSCs as a novel cell therapy in future clinical applications for delayed and non-healing wounds associated with CLTI.