SBIR Phase I: A novel class of molecular vehicles for the targeted and precise integration of specified genetic information into the genomes of host cells and organisms

这个小企业创新研究（SBIR）第一阶段项目的更广泛影响是推进遗传信息（遗传有效载荷）人工整合到动物细胞、微生物和植物的基因组中。一类新型分子载体（DNA供体）与已建立的用于基因编辑和基因组工程的CRISPR（聚集短间隔回文重复序列）系统相结合，可以以前所未有的效率传递遗传有效载荷。新的有效载荷交付技术有望实现许多新兴和创新应用。这项技术以较低的成本为最先进的方法提供了更安全的替代方案。非治疗应用将使细胞系和转基因动物的有效产生。在植物生物技术方面，新的DNA供体将允许只进行一轮基因组修改就能进行复杂的基因工程，从而提高作物产量和农业产量。拟议的项目为DNA供体提供了一个关键的创新，用于通过CRISPR转移基因有效载荷进行靶向整合。尽管crispr介导的基因破坏（敲除）非常有效，但将转染的DNA供体整合到靶向整合（敲入）中通常是有限的。现有的商业解决方案主要集中在小基因大小的有效载荷上，但迫切需要一种创新的解决方案，使携带多个基因或整个遗传回路的更大有效载荷集成在一起。本提案中的工作将证明基因转移和编辑技术在治疗相关细胞类型（如干细胞和原代免疫细胞）中的广泛适用性。将通过比较其整合效率、整合精度以及与传统DNA供体分子和基于病毒的递送系统的细胞毒性来评估新型供体载体的性能。此外，该项目将展示生产大量新DNA供体的能力，从而大大促进其在细胞治疗中的应用，并大大降低其总体成本。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 of this Small Business Innovation Research (SBIR) Phase I project is to advance artificial integration of genetic information (genetic payloads) into the genomes of animal cells, microorganisms, and plants. A novel class of molecular vehicles (DNA donors) allows an unprecedented efficiency in delivering genetic payloads when combined with the established CRISPR (clustered short interspaced palindromic repeats) system for gene editing and genome engineering. The new payload delivery technology is expected to enable many emergent and innovative applications. This technology provides a safer alternative to the state-of-the-art method at lower cost. Non-therapeutic applications will enable the efficient generation of cell lines and transgenic animals. In plant biotechnology, the new DNA donors will allow complex genetic engineering with only one round of genome modification, improving crop yields and agricultural production.The proposed project provides a key innovation to the DNA donors that are used for transferring genetic payloads for targeted integration via CRISPR. Whereas CRISPR-mediated gene disruption (knock-out) is very efficient, integration of transfected DNA donors for targeted integration (knock-in) is generally limited. Existing commercial solutions are focused on small gene-sized payloads, but an innovative solution that enables the integration of larger payloads carrying several genes or entire genetic circuits is critically needed. The work in this proposal will demonstrate the broad applicability of the gene transfer and editing technology in a subset of therapeutically relevant cell types, such as stem cells and primary immune cells. The performance of the novel donor vehicles will be assessed by comparing their integration efficiency and precision of integration and cell toxicity to conventional DNA donor molecules and virus-based delivery systems. In addition, the project will demonstrate the ability to produce large quantities of the new DNA donors, thus greatly facilitating their application in cell therapies and dramatically reducing their overall cost.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.