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

这项小企业创新研究（SBIR）第一阶段项目的更广泛影响/商业潜力是利用CRISPR为遗传性疾病患者带来治疗性基因编辑疗法。基于crispr的治疗方法有可能成为下一代治疗方法，特别是对于遗传性疾病，因为它们能够以序列特异性切割DNA。然而，这种类型的靶向基因组编辑尚未用现有方法成功地证明用于人类治疗。将基于crispr的基因编辑转化为临床仍然是一个重要的未满足需求，将基于crispr的基因编辑转化为治疗方法最困难的方面是缺乏安全有效的靶向组织递送方法。现有的基于病毒的递送系统存在一些局限性，包括免疫原性、预先存在的抗体、广泛的趋向性、脱靶效应、受限制的DNA货物包装能力和制造挑战。因此，使用合成材料的非病毒方法正被广泛研究作为潜在的替代品。开发能够有效地将CRISPR成分递送到目标组织的非病毒递送载体将提高广泛使用基于CRISPR的治疗方法治疗许多遗传疾病的能力。这个SBIR一期项目的智力优势在于开发一种CRISPR酶Cas 12a （Cpf1）的非病毒递送系统，用于递送到目标肌肉组织，并有可能开发一种治疗杜氏肌营养不良症（DMD）的新型基因编辑治疗方法。该方法涉及专有的crispr纳米颗粒，由肽PEG-PAsp（DET）与Cpf1 RNP复合物组成，具有良好的生物相容性和高基因编辑效率，具有在GMP下生产和扩大规模用于临床试验的潜力。初步数据表明，PEG-PAsp（DET）与Cpf1 RNP复合物可以有效地将Cpf1 RNP传递到肌肉组织，并通过突变删除23外显子诱导肌营养不良蛋白的表达。在此建议下，具体的实验将侧重于提高crispr纳米颗粒的生物相容性、稳定性和肌肉靶向能力。实验计划是合成多种肽- peg - pasp (DET)，然后在原代成肌细胞和报告小鼠系统中进行筛选。这项使用crispr纳米颗粒的技术将成为首个通过静脉注射同时递送Cpf1蛋白和gRNA并实现基因编辑的运载工具。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.