Krabbe disease therapy integrating gene transfer with lectin-enhanced enzyme delivery to treat pathologies of the CNS

Krabbe病(球状细胞白质营养不良)是一种罕见的遗传性疾病，由大脑中的 溶酶体酶半乳糖神经酰胺酶(GALC)导致保护膜(髓鞘)的破坏 大脑和整个神经系统中的神经细胞。这种致命的神经退行性疾病会影响 在美国，大约每10万人中就有1人。在大多数情况下，婴儿在6个月前出现克拉贝症状 年龄，通常导致2岁前死亡。骨髓移植是目前唯一被批准的治疗方法 Krabbe患者的选择，但只会减缓疾病的进展，而且只有在症状前期启动时才会 孩子们。最近的临床前研究主要集中在用腺相关病毒(Aav)进行基因转移。 用于在Krabbe病的Twitcher小鼠模型中长期表达正常的GALC酶。 尽管这种方法很有希望，但在向危重病患者分发有效治疗剂量方面仍存在不足。 大脑和神经系统的一部分。BioStrategy LC已经开发出一种基于凝集素的治疗性递送 模块(RTB)，使融合的溶酶体酶能够有效地靶向中枢神经系统并治疗神经病 每周输注ERT后的症状，如在几个溶酶体疾病动物模型中所显示的。 在这个SBIR项目中，我们建议融合AAV介导的基因转移技术的好处，这些技术 用RTB递送技术提供持续的长期体内酶生产，以确保 克雷布进行性神经变性的有效治疗。GALC与GALC融合的基因表达盒 BioStrategy的RTB凝集素将被包装成重组腺相关病毒用于生产和 随后给抽搐小鼠灌胃。对小鼠神经系统疾病进展和寿命的影响 接受AAV载体GALC-RTB融合的小鼠将与未治疗和注射的小鼠进行比较 单独使用AAV载体的GALC。本次第一阶段可行性研究的具体目标是：1)构建GALC-RTB AAV 载体并证明其在哺乳动物细胞中产生全功能GALC-RTB融合产物的能力 比较AAV介导的GALC-RTB基因转移与GALC-RTB基因转移对GALC的治疗效果 在Krabbe小鼠模型中，通过脑部和周围神经病理的寿命和成像来衡量。 第一阶段的里程碑将表明，我们的凝集素融合，GALC-rtb，可以在Krabbe twitcher中表达 AAV基因转移模型及GALC-RTB对中枢神经系统难治细胞的靶向性研究 和PNS组织在抽动者(GALC-/-)小鼠中，促进酶的生物分布和寿命。基于这些 概念验证结果，第二阶段研究将评估药物剂量水平、时间和路线的影响 关于长期治疗的有效性和安全性的管理，旨在进一步将该产品转移到IND。 该项目的长期目标是开发并将这种新型的“递送增强型”AAV基因疗法带到 克拉布病人。在这里建立的可行性也将支持扩大“rtb凝集素融合”基因。 治疗其他有明显中枢神经系统和三叉神经系统组织损害的罕见疾病。

Krabbe disease (globoid cell leukodystrophy) is a rare inherited disorder caused by a deficiency in the lysosomal enzyme galactosylceramidase (GALC) leading to destruction of the protective coating (myelin) of nerve cells in the brain and throughout the nervous system. This fatal neurodegenerative disease affects about 1 in 100,000 people in the US. In most cases, Krabbe symptoms develop in infants before 6 months of age, often leading to death by age 2. Bone marrow transplantation is currently the only approved treatment option for Krabbe patients, but only slows disease progression, and only when initiated in pre-symptomatic children. More recent preclinical research has focused on gene transfer utilizing adeno-associated virus (AAV) for long term expression of the normal GALC enzyme in the “twitcher” mouse model of Krabbe disease. Although promising, this approach still shows deficiencies in distributing effective therapeutic doses to critical parts of the brain and nervous system. BioStrategies LC has developed a lectin-based therapeutic delivery module (RTB) that enables fused lysosomal enzymes to effectively target the CNS and treat neurological symptoms following weekly ERT infusions, as demonstrated in several lysosomal disease animal models. In this SBIR project, we propose to merge the benefits of AAV-mediated gene transfer technologies that provide continuous long-term in vivo enzyme production with those of the RTB delivery technology to ensure effective treatment of Krabbe’s progressive neurodegeneration. Gene expression cassettes fusing GALC with BioStrategies’ RTB lectin will be packaged into recombinant adeno-associated virus for production and subsequent administration to twitcher mice. Impacts on neurological disease progression and lifespan of mice receiving AAV-vectored GALC-RTB fusions will be compared with untreated mice and those administered AAV-vectored GALC alone. Specific aims of this Phase I feasibility study are 1) to construct GALC-RTB AAV vectors and demonstrate their ability to produce fully functional GALC-RTB fusion product in mammalian cells and mice; and 2) to compare therapeutic efficacy of AAV-mediated gene transfer of GALC-RTB versus GALC in the Krabbe mouse model as measured by lifespan and imaging of brain and peripheral nerve pathologies. Phase I milestones will show that our lectin fusion, GALC-RTB, can be expressed in the Krabbe twitcher model via AAV gene transfer and demonstrate targeting of expressed GALC-RTB to hard-to-treat cells of CNS and PNS tissue in twitcher (GALC-/-) mice, enhancing enzyme biodistribution and lifespan. Based on these proof-of concept results, Phase II studies will assess the effects of dosage levels, timing, and routes of drug administration on efficacy and safety of long-term treatment aimed at further moving this product to an IND. The long-term goal of this project is to develop and bring this novel “delivery-enhanced” AAV gene therapy to Krabbe patients. The feasibility established here will also support expanding the “RTB lectin-fused” gene therapy treatment to other rare diseases having significant CNS and PNS tissue impairment.