Autologous TGF-B-Modified HSC for Repair of Vasodegenerative Diabetic Retinopathy

自体 TGF-B 修饰的 HSC 用于修复血管退行性糖尿病视网膜病变

• 批准号: 7745244
• 负责人: Stephen Hollis Bartelmez
• 金额: $ 32.67万
• 依托单位: BETASTEM THERAPUETICS, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7745244
• 关键词: Address; Adoptive Transfer; Adult; Age; Area; Autologous; Blindness; Blood; Blood Cells; Blood Vessels; Blood capillaries; Bone Marrow; Bone Marrow Stem Cell; CD34 gene; CXCR4 gene; Cell physiology; Cells; Chemotaxis; Clinical Treatment; Clinical Trials; Data; Diabetes Mellitus; Diabetic Retinopathy; Disease; Edema; Engraftment; Ensure; Environment; Eye; Florida; Generations; Goals; Grant; Growth; Health; Hematopoietic stem cells; Hemorrhage; Homing; Human; Ischemia; Laboratories; Lead; Legal patent; Measures; Modeling; Molecular; Mus; Neurons; Neuropathy; Non-Insulin-Dependent Diabetes Mellitus; Output; Oxygen; Pathology; Patients; Peripheral Blood Stem Cell; Pharmaceutical Preparations; Pharmacology; Phase; Prevention; Reperfusion Injury; Retina; Retinal; Retinal Diseases; Retinal Neovascularization; SCID Mice; Safety; Series; Staging; Stem cells; Streptozocin; Techniques; Testing; Therapeutic; Time; Transforming Growth Factor beta; Tube; Universities; Vision; Work; base; capillary; capillary bed; chemokine; diabetic; diabetic patient; effective therapy; end stage disease; human TGFB1 protein; in vitro Assay; injured; macular edema; non-diabetic; novel; novel strategies; paracrine; phosphorodiamidate morpholino oligomer; prevent; public health relevance; reconstitution; repaired; research study; retina blood vessel structure; senescence; stem cell therapy

DESCRIPTION (provided by applicant): Diabetic retinopathy induces retinal vascular endothelial damage resulting in ischemia, edema, and hemorrhage. Current available treatments for diabetic retinopathy are focused on end-stages of the disease. Importantly, these therapies do not address the primary pathology of retinal neurovascular degeneration during diabetes that precedes pre-retinal neovascularization and diabetic macular edema. Fresh perspective on the cellular mechanisms of DR could lead to novel and much more effective prevention / reversal strategies. We describe here a new approach to target early intermediate stages of vasodegeneration using a stem cells therapy to enhance vessel repair, reverse ischemia and prevent progression to late, sight -threatening stages of diabetic retinopathy. For patients with loss of retinal microvasculature, measures to preserve surviving vasculature and revascularize defunct capillary beds could extend the lifetime of the neuronal cells, reduce output of vasoactive and neuropathic agents and ensure retention of serviceable vision. We have developed a novel strategy to enhance endothelial progenitor cells (EPC) function both in health and in diseases such as diabetes. We recently demonstrated that a transient blockade (2-4 days) of endogenous transforming growth factor- beta type 1 (TGF-b1) in murine and human hematopoietic stem cells (HSC and EPCs ) accelerates key functions of these cells including 1) bone marrow engraftment, 2) reducing the number of HSC needed for long-term reconstitution, 3) increasing CXCR-4 expression enhancing the responsiveness of these cells to stromal derived factor (SDF)-1, a key chemokine for EPC chemotaxis including increased NO generation following SDF-1 exposure cells. And 4) increases the reparative function of human diabetic CD34 EPC in SCID mice following ischemia/ reperfusion injury. Overall Hypothesis: Transient blockade of endogenous transforming growth factor-beta type 1 (TGF-b1) using antisense phosphorodiamidate morpholino oligomers (PMO) to TGF-b1 in diabetic CD34+HSC will potentiate their ability to repair retinal vessels in diabetes. To test our hypothesis we put for the following Milestones Phase I: AIM 1: Our hypothesis predicts that transient TGF-2 blockade will increase homing ability and proliferation rate of CD34+ HSC in damaged retinal vessels diabetic patients with retinopathy. While this effect in the HSC is transient (2-4 days), it is sufficient to result in enhanced reparative function of these cells. The molecular mechanism of this effect will be examined in vitro by assays to assess changes in proliferation, capillary tube formation and rate of senescence. AIM 2: Our hypothesis predicts that TGF-b blockade enhances the reparative function of CD34+ cells by facilitating their homing to the injured retina. Either direct incorporation into blood vessels in the area of ischemia or paracrine expression of growth promoting factors could enhance the observed repair. To test this hypothesis we will use the adoptive transfer technique to deliver anti-TGF-2 PMO-modified CD34+ cells from the blood or bone marrow of patients with type II diabetes to SCID mice that have 1) undergone an ischemia/reperfusion injury model that results in vasodegeneration of retinal capillaries or 2) been made diabetic with STZ. Control CD34+ cells will be from non-diabetic age matched subjects. These proposed collaborative studies between BetaStem Therapeutics and Dr. Maria Grant's laboratory (University of Florida, Department of Pharmacology and Therapeutics) are based on the series of complementary findings summarized above. Much of the studies presented in the preliminary data section are either published1 or under review. 2 The approach proposed has been IP protected by two issued US Patents as well as pending patents. Our Phase 2 proposal will focus on clinical trials that will be based on pre- treatment of autologous diabetic CD34+ cells with anti-TGF-b1 PMOs that specifically block endogenous TGF- b1 in CD34+ cells. We hypothesize that transient TGF-b1 blockade will restore and enhance the reparative function of dysfunctional diabetic EPC and these modified EPCs will serve as a treatment for the vasodegenerative phase of diabetic retinopathy. PUBLIC HEALTH RELEVANCE: Our long term goal is to develop an efficient, safe clinical treatment for diabetic retinopathy using stem cells from the patient's bone marrow or blood that have been activated to repair damaged vessels in the eye. Despite advances in our understanding of how diabetic retinopathy occurs, no effective treatment exists to reverse the retinal blood vessel damage and the vision loss resulting from lack of blood/oxygen supply to the retina. Diabetic retinopathy remains the leading cause of irreversible blindness among working-age adults. We have shown that, over time, the diabetic cellular environment markedly inhibits the ability of their stem cells to repair damaged blood vessels. However, we now show that if these stem cells are treated temporarily with a drug that blocks an important regulator of stem cell activation (i.e., transforming growth factor-beta, TGF-2) vessel repair is greatly enhanced. In this proposal, we plan to test the repair capacity of stem cells obtained from diabetic patients to those from non- diabetic patients since our preliminary data suggest that stem cells from bone marrow of non-diabetic normal donors may repair better than stem cells from peripheral blood. Next, we will conduct a series of experiments to better understand the cellular mechanisms that result in the observed more efficient vascular repair. We also will test the long term safety of introducing stems cells into the eye. Once we complete these proposed studies, we will be able to plan a clinical trial to treat patients with diabetic retinopathy using their own stem cells. If this treatment proves to be safe and effective, it will be the first "cure" for diabetic retinopathy.

描述（由申请人提供）：糖尿病视网膜病变引起视网膜血管内皮损伤，导致缺血、水肿和出血。目前可用的糖尿病视网膜病变治疗主要集中在疾病的终末期。重要的是，这些疗法不能解决糖尿病视网膜神经血管变性的主要病理，这种变性发生在视网膜前新生血管形成和糖尿病黄斑水肿之前。对DR的细胞机制的新视角可能导致新的和更有效的预防/逆转策略。我们在这里描述了一种新的方法，针对早期中期血管变性使用干细胞治疗，以增强血管修复，逆转缺血和防止进展到晚期，视力威胁阶段的糖尿病视网膜病变。对于视网膜微血管丧失的患者，采取措施保留残存的血管和重建已失效的毛细血管床，可以延长神经元细胞的寿命，减少血管活性和神经性药物的输出，并确保保留可用的视力。我们已经开发了一种新的策略来增强内皮祖细胞（EPC）在健康和糖尿病等疾病中的功能。我们最近证明，小鼠和人造血干细胞（HSC和EPCs）中内源性转化生长因子- β 1型（TGF-b1）的短暂阻断（2-4天）加速了这些细胞的关键功能，包括1)骨髓移植，2)减少长期重建所需的HSC数量，3)增加CXCR-4的表达，增强了这些细胞对基质衍生因子(SDF)-1的反应性。一个关键的趋化因子，包括SDF-1暴露后细胞增加NO生成。4)增加人糖尿病CD34 EPC在SCID小鼠缺血再灌注损伤后的修复功能。总体假设：在糖尿病CD34+HSC中，使用反义磷酸二酯morpholino寡聚物（PMO）对TGF-b1的短暂阻断内源性转化生长因子- 1型（TGF-b1）将增强其修复糖尿病视网膜血管的能力。为了验证我们的假设，我们提出了以下里程碑I期：AIM 1：我们的假设预测，暂时性TGF-2阻断将增加受损视网膜血管糖尿病视网膜病变患者CD34+ HSC的归巢能力和增殖率。虽然这种作用在HSC中是短暂的（2-4天），但足以增强这些细胞的修复功能。这种作用的分子机制将通过体外实验来评估增殖、毛细血管形成和衰老速度的变化。AIM 2：我们的假设预测，TGF-b阻断通过促进CD34+细胞归巢到受损视网膜，增强了CD34+细胞的修复功能。生长促进因子直接掺入缺血区血管或旁分泌表达均可促进观察到的修复。为了验证这一假设，我们将使用过继性转移技术，将抗tgf -2 pmo修饰的CD34+细胞从II型糖尿病患者的血液或骨髓中传递给SCID小鼠，这些小鼠经历了缺血/再灌注损伤模型，导致视网膜毛细血管变性，或2)患有STZ的糖尿病。对照CD34+细胞将来自非糖尿病年龄匹配的受试者。BetaStem Therapeutics公司和Maria Grant博士的实验室（佛罗里达大学药理学和治疗学系）提出的这些合作研究是基于上述总结的一系列互补发现。初步数据部分提出的许多研究要么已发表，要么正在审查中。所提出的方法已受到两项已发布的美国专利和正在申请的专利的知识产权保护。我们的二期计划将侧重于临床试验，这些临床试验将基于使用抗TGF-b1 PMOs对自体糖尿病CD34+细胞进行预处理，该PMOs可特异性阻断CD34+细胞中的内源性TGF-b1。我们假设短暂的TGF-b1阻断将恢复和增强功能失调的糖尿病EPCs的修复功能，这些修饰的EPCs将作为糖尿病视网膜病变血管退行性阶段的治疗方法。公共卫生相关性：我们的长期目标是开发一种有效、安全的糖尿病视网膜病变临床治疗方法，使用来自患者骨髓或血液的干细胞，激活修复眼部受损血管。尽管我们对糖尿病视网膜病变如何发生的了解有所进展，但目前还没有有效的治疗方法来逆转视网膜血管损伤和因视网膜缺乏血液/氧气供应而导致的视力丧失。糖尿病视网膜病变仍然是导致工作年龄成年人不可逆转失明的主要原因。我们已经证明，随着时间的推移，糖尿病细胞环境明显抑制了他们的干细胞修复受损血管的能力。然而，我们现在表明，如果用一种阻断干细胞激活的重要调节因子（即转化生长因子- β， TGF-2）的药物暂时治疗这些干细胞，血管修复将大大增强。在这个提议中，我们计划测试从糖尿病患者身上获得的干细胞与非糖尿病患者身上获得的干细胞的修复能力，因为我们的初步数据表明，来自非糖尿病正常供者骨髓的干细胞可能比来自外周血的干细胞修复得更好。接下来，我们将进行一系列实验，以更好地了解导致观察到的更有效的血管修复的细胞机制。我们还将测试将干细胞引入眼睛的长期安全性。一旦我们完成这些拟议的研究，我们将能够计划一项临床试验，使用患者自己的干细胞治疗糖尿病视网膜病变。如果这种治疗被证明是安全有效的，它将是第一个“治愈”糖尿病视网膜病变的方法。