SBIR Phase I: A block copolymer delivery system for the Cpf1 ribonucleoprotein

这个小企业创新研究(SBIR)第一阶段项目的更广泛的影响/商业潜力是使用CRISPR为遗传病患者带来根治性的基因编辑疗法。基于CRISPR的疗法具有成为下一代疗法的潜力，特别是对于遗传病，因为它们能够切割具有序列特异性的DNA。然而，这种有针对性的基因组编辑还没有用现有的方法成功地用于人类治疗。将基于CRISPR的基因编辑转化到临床仍然是一个重要的未得到满足的需求，将基于CRISPR的基因编辑转化为治疗最困难的方面是缺乏安全有效的靶向组织的传递方法。现有的基于病毒的递送系统具有局限性，包括免疫原性、先前存在的针对它们的抗体、广泛的趋向性、非靶标效应、有限的DNA货物包装能力以及制造挑战。因此，使用合成材料的非病毒方法正被广泛研究为潜在的替代品。开发能够有效地将CRISPR组分输送到靶组织的非病毒递送载体将提高广泛使用基于CRISPR的疗法治疗多种遗传病的能力。这项SBIR第一阶段项目的智力优势是为CRISPR酶Cas 12A(Cpf1)开发一种非病毒递送系统，用于递送到靶肌肉组织，并有可能开发一种治疗Duchenne肌营养不良症(DMD)的新型基因编辑疗法。该方法涉及专利CRISPR纳米颗粒，它由与Cpf1 RNP络合的多肽PEG-PAsp(Det)组成，具有良好的生物兼容性和高基因编辑效率，有可能在GMP下制造和放大用于临床试验。初步研究表明，与Cpf1 RNP结合的聚乙二醇-PAsp(Det)能有效地将Cpf1 RNP运送到肌肉组织，并通过突变缺失外显子23来诱导dystrophin蛋白的表达。根据这一建议，具体的实验将集中在提高CRISPR纳米颗粒的生物相容性、稳定性和肌肉靶向能力上。实验计划是合成各种多肽-聚乙二醇-聚天冬氨酸(DET)，然后在原代成肌细胞和报告鼠系统中进行筛选。这项使用CRISPR纳米颗粒的技术将是第一个可以通过静脉注射同时传递Cpf1蛋白和gRNA并实现基因编辑的载体。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project is to bring curative gene editing therapies using CRISPR to patients with genetic diseases. CRISPR-based therapeutics have the potential to be next generation therapeutics, particularly for genetic diseases due to their ability to cut DNA with sequence specificity. However, this type of targeted genome editing has not yet been successfully demonstrated for human therapeutics with existing methods. The translation of CRISPR-based gene editing to the clinic remains a significant unmet need and the most difficult aspect of translating CRISPR-based gene editing into therapeutics is the lack of safe and effective delivery methods to the target the tissues. Existing viral-based delivery systems have limitations that include immunogenicity, pre-existing antibodies against them, broad tropism, off-target effects, restricted DNA cargo packaging capacity, and manufacturing challenges. As a result, non-viral methods that employ synthetic materials are being widely investigated as potential alternatives. Developing non-viral delivery vehicles that can effectively deliver CRISPR components to target tissues will improve the ability to broadly use CRISPR-based therapeutics for many genetic diseases.The intellectual merit of this SBIR Phase I project is to develop a non-viral delivery system for the CRISPR enzyme Cas 12a (Cpf1) for delivery to target muscle tissue, and, potentially, enable the development of a novel gene editing treatment for Duchenne muscular dystrophy (DMD). The approach involves proprietary CRISPR-nanoparticles that are composed of peptide PEG-PAsp(DET) complexed to Cpf1 RNP, which possess good biocompatibility and high gene editing efficiency with the potential to manufacture and scale up under GMP for use in clinical trials. Preliminary data demonstrate that PEG-PAsp(DET) complexed to Cpf1 RNP can efficiently deliver Cpf1 RNP to the muscle tissue and can induce the expression of the dystrophin protein by deleting exon23 with a mutation. Under this proposal, the specific experiments will focus on improving the biocompatibility, stability, and muscle-targeting ability of the CRISPR-nanoparticles. The experimental plan is to synthesize various Peptide-PEG-PAsp(DET), followed by screening in primary myoblasts and reporter mouse system. This technology using CRISPR-nanoparticles will be the first example of a delivery vehicle that can simultaneously deliver Cpf1 protein and gRNA via intravenous injection and achieve gene editing.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.