Automated discovery of noncovalent inhibitors of SARS-CoV-2 main protease by consensus Deep Docking of 40 billion small molecules.

Recent explosive growth of 'make-on-demand' chemical libraries brought unprecedented opportunities but also significant challenges to the field of computer-aided drug discovery. To address this expansion of the accessible chemical universe, molecular docking needs to accurately rank billions of chemical structures, calling for the development of automated hit-selecting protocols to minimize human intervention and error. Herein, we report the development of an artificial intelligence-driven virtual screening pipeline that utilizes Deep Docking with Autodock GPU, Glide SP, FRED, ICM and QuickVina2 programs to screen 40 billion molecules against SARS-CoV-2 main protease (Mpro). This campaign returned a significant number of experimentally confirmed inhibitors of Mpro enzyme, and also enabled to benchmark the performance of twenty-eight hit-selecting strategies of various degrees of stringency and automation. These findings provide new starting scaffolds for hit-to-lead optimization campaigns against Mpro and encourage the development of fully automated end-to-end drug discovery protocols integrating machine learning and human expertise.

Mammalian artificial chromosomes and clinical applications for genetic modification of stem cells: an overview.

Modifying multipotent, self-renewing human stem cells with mammalian artificial chromosomes (MACs), present a promising clinical strategy for numerous diseases, especially ex vivo cell therapies that can benefit from constitutive or overexpression of therapeutic gene(s). MACs are nonintegrating, autonomously replicating, with the capacity to carry large cDNA or genomic sequences, which in turn enable potentially prolonged, safe, and regulated therapeutic transgene expression, and render MACs as attractive genetic vectors for "gene replacement" or for controlling differentiation pathways in progenitor cells. The status quo is that the most versatile target cell would be one that was pluripotent and self-renewing to address multiple disease target cell types, thus making multilineage stem cells, such as adult derived early progenitor cells and embryonic stem cells, as attractive universal host cells. We will describe the progress of MAC technologies, the subsequent modifications of stem cells, and discuss the establishment of MAC platform stem cell lines to facilitate proof-of-principle studies and preclinical development.

Transgene expression after stable transfer of a mammalian artificial chromosome into human hematopoietic cells.

OBJECTIVE: The transfer of mammalian artificial chromosomes (MACs) to hematopoietic stem and progenitor cells (HSPCs) presents a promising new strategy for ex vivo gene therapy that alleviates numerous concerns surrounding viral transduction along with a unique platform for the systematic study of stem cell biology and fate. Here we report the transfer of a satellite DNA-based artificial chromosome (an ACE), made in mouse cells, into human cord blood hematopoietic cells. MATERIALS AND METHODS: A GFP-Zeo-ACE encoding the genes for humanized Renilla green fluorescence protein (hrGFP) and zeomycin resistance (zeo) was transferred into CD34 positively selected cord blood cells using cationic reagents. RESULTS: Post ACE transfer, CFU-GM-derived colonies were generated in methylcellulose in the presence or absence of bleomycin. Bleomycin-resistant cells expressed GFP and contained intact autonomous ACEs, as demonstrated by fluorescent in situ hybridization. Moreover, when the cells from these plates were replated in methylcellulose, we observed secondary bleomycin-resistant CFU-GM-derived colonies, demonstrating stable chromosome retention and transgene function in a CFU-GM progenitor. CONCLUSION: To our knowledge this is the first report demonstrating the transfer of a mammalian artificial chromosome and the stable expression of an encoded transgene in human hematopoietic cells.

Development of mammalian artificial chromosomes for the treatment of genetic diseases: Sandhoff and Krabbe diseases.

Mammalian artificial chromosomes (MACs) are being developed as alternatives to viral vectors for gene therapy applications, as they allow for the introduction of large payloads of genetic information in a non-integrating, autonomously replicating format. One class of MACs, the satellite DNA-based artificial chromosome expression vehicle (ACE), is uniquely suited for gene therapy applications, in that it can be generated denovo in cells, along with being easily purified and readily transferred into a variety of recipient cell lines and primary cells. To facilitate the rapid engineering of ACEs, the ACE System was developed, permitting the efficient and reproducible loading of pre-existing ACEs with DNA sequences and/or target gene(s). As a result, the ACE System and ACEs are unique and versatile platforms for ex vivo gene therapy strategies that circumvent and alleviate existing safety and delivery limitations surrounding conventional gene therapy vectors. This review will focus on the status of MAC technologies and, in particular, the application of the ACE System towards an ex vivo gene therapy treatment of lysosomal storage diseases, specifically Sandhoff (MIM #268800) and Krabbe (MIM #245200) diseases.

A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy.

Mammalian artificial chromosomes (MACs) provide a means to introduce large payloads of genetic information into the cell in an autonomously replicating, non-integrating format. Unique among MACs, the mammalian satellite DNA-based Artificial Chromosome Expression (ACE) can be reproducibly generated de novo in cell lines of different species and readily purified from the host cells' chromosomes. Purified mammalian ACEs can then be re-introduced into a variety of recipient cell lines where they have been stably maintained for extended periods in the absence of selective pressure. In order to extend the utility of ACEs, we have established the ACE System, a versatile and flexible platform for the reliable engineering of ACEs. The ACE System includes a Platform ACE, containing >50 recombination acceptor sites, that can carry single or multiple copies of genes of interest using specially designed targeting vectors (ATV) and a site-specific integrase (ACE Integrase). Using this approach, specific loading of one or two gene targets has been achieved in LMTK(-) and CHO cells. The use of the ACE System for biological engineering of eukaryotic cells, including mammalian cells, with applications in biopharmaceutical production, transgenesis and gene-based cell therapy is discussed.