Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.
 
Key Points:

• SLEEK can generate multiplex-edited allogeneic T cells with near homogeneity.
• SLEEK can be used to insert up to four transgene cargos in tandem in a single knock-in cassette.
• SLEEK offers tunable expression of the transgene cargo through knock-in at other essential genes each with their own intrinsic endogenous promoter strengths.

Employing a metagenomic search, we identified a family of miniature CRISPR-Cas type V-L systems capable of RNA-guided dsDNA target cleavage without a tracrRNA. A bacterial depletion screen revealed several active systems which were subsequently shown to have low-level editing in mammalian cells. Among the systems with activity in mammalian cells, we selected a 745aa nuclease effector as a candidate for engineering. An unbiased mutational scanning approach was applied to identify single substitutions that increase indel activity in mammalian cells. Indel-enhancing single substitutions were then screened in combination, with a combinatorially engineered variant demonstrating indel editing activity comparable to SpCas9 in HEK293T cells and exceeding 60% in primary human hepatocytes. Structure determination via cryo-EM revealed the domain organization and nuclease mechanism of the ternary complex. Due to its small size and robust editing levels, this novel miniature CRISPR-Cas type V-L system is an attractive therapeutic candidate for single- or dual-guide excision via all-in-one AAV delivery. 
 
Key Points:
Small size coupled with potent mammalian activity make the combinatorial engineered variant of a miniature type V-L CRISPR-Cas system an attractive candidate for therapeutic genome editing.

Cell-based immunotherapy has become the new-generation cancer medicine, and “off-the-shelf” cell products that can be manufactured at large scale and distributed readily to treat patients are necessary. Invariant natural killer T (iNKT) cells are ideal cell carriers for developing allogeneic cell therapy because they are powerful immune cells targeting cancers without graft-versus-host disease (GvHD) risk. However, healthy donor blood contains extremely low numbers of endogenous iNKT cells. By combining hematopoietic stem cell (HSC) gene engineering and in vitro differentiation, we have generated human allogeneic HSC-engineered iNKT (AlloHSC-iNKT) cells at high yield and purity; these cells closely resemble endogenous iNKT cells, effectively target tumor cells using multiple mechanisms, and exhibit high safety and low immunogenicity. These cells can be further engineered with chimeric antigen receptor (CAR) to enhance tumor targeting or/and gene edited to ablate surface human leukocyte antigen (HLA) molecules and further reduce immunogenicity. Collectively, these preclinical studies demonstrate the feasibility and cancer therapy potential of AlloHSC-iNKT cell products and lay a foundation for their translational and clinical development.
 
Key Points:

• Allogeneic HSC-iNKT cells are generated in vitro at high yield and purity
• Allogeneic HSC-iNKT cells effectively target tumor cells using multiple mechanisms
• Allogeneic HSC-iNKT cells exhibit high safety and low immunogenicity
• A preclinical study demonstrated feasibility, safety, and cancer therapy potential

Phosphorylation of α-synuclein at the Serine-129 site (α-syn Ser129P) is an established pathologic hallmark of synucleinopathies and a therapeutic target. In physiologic states, only a tiny fraction of α-syn is phosphorylated at this site, and most studies have focused on the pathologic roles of this post-translational modification. Dr. Parra-Rivas found that unlike wild-type (WT) α-syn, widely expressed throughout the brain, the overall pattern of α-syn Ser129P is restricted, suggesting intrinsic regulation. Surprisingly, preventing Ser129P blocked activity-dependent synaptic attenuation by α-syn – thought to reflect its normal function. Exploring mechanisms, Dr. Parra-Rivas found that neuronal activity augments Ser129P, which triggers protein-protein interactions necessary for mediating α-syn function at the synapse. AlphaFold2-driven modeling and membrane-binding simulations suggest a scenario where Ser129P induces conformational changes that facilitates interactions with binding partners. Dr. Parra-Rivas' experiments offer a new conceptual platform for investigating the role of Ser129 in synucleinopathies, with implications for gene editing therapies. Together with Drs. Madhivanan and Roy he continues investigating the physiological roles of α-synuclein at synapses using CRISPR-based strategies.

Key Points:

• Phosphorylation of α-syn at the Ser129-site (α-syn Ser129P) is a pathologic hallmark of synucleinopathies that is often synonymously associated with disease, but our studies suggest that this post-translational modification has a physiologic role.
• α-syn is widely expressed in synapses throughout the brain, whereas Ser129P is only seen in a subset of brain regions, and this phosphorylation augments the synaptic targeting of α-syn.
• Ser129P facilitates protein-protein interactions at the synapse and functions as a brake on activity-dependent synaptic vesicle recycling.
• Our data reveal a cascade of molecular events by which Ser129P of α-syn regulates synaptic function and offers a new basis for understanding the role of this post-translational modification in pathophysiology.

The use of adeno-associated viruses (AAV) as gene delivery vectors has vast potential for the treatment of many severe human diseases. A small group of intensively studied AAV capsids have been propelled into pre-clinical and clinical use, and more recently, marketed products; however, many less-studied, naturally occurring capsids may also have desirable properties (e.g. potency differences, tissue tropism, reduced immunogenicity, etc.) that have yet to be thoroughly described. Within this under-studied group of capsids is AAV7, a potentially therapeutic capsid for which tropism studies are limited, and direct, head-to-head comparisons with well-described capsids have not been performed. We sought to characterize the biodistribution of this capsid more fully by several methods including quantitative PCR, immunofluorescence (IF) and immunohistochemistry (IHC), and whole-body imaging via cryofluorescence tomography (CFT) as an unbiased method to look for novel regions of transduction.
CFT of whole mice was performed three weeks after IV administration of AAV7 or AAV9 carrying an EGFP transgene driven by CAG, or co-administration of AAV7-EGFP and AAV9-tdTomato. This was compared to traditional biodistribution analysis of vector genome copies and expressed RNA transcripts. Superiority of AAV9 in mouse skeletal muscle was shown over AAV7 and was confirmed via CFT and IF. Cardiac transduction by AAV7 trended higher than AAV9 in all studies. AAV7 and AAV9 both transduce the mouse brain with equal efficiency according to PCR-based biodistribution data; however, by CFT and IHC, cell type differences were found within the brain, with AAV7 being localized mostly around blood vessels within the brain, and AAV9 widely transducing astrocytes and neurons. A surprising finding from CFT was the presence of fluorescence in facial structures, some of it highly concentrated, for both AAV7 and AAV9. As this is not typically an area sampled by traditional biodistribution methods, this finding has been largely overlooked. In particular, the mandible showed the highest intensity of EGFP fluorescence of any other tissue for both AAV7 and AAV9. The incisor also had measurable EGFP fluorescence. As the mouse mandible is surrounded by muscle tissue that likely was transduced, we conducted an IHC study to further ascertain detailed information about the transduction seen in and around mouse facial structures and investigate tropism differences between AAV7 and AAV9. Surprisingly, strong transgene expression was observed throughout the head, as verified by IHC, including regions of the cranial sinuses, teeth, and mandible for both AAV7 and AAV9, an attribute that has not been previously evaluated via traditional biodistribution methods. These data will help build a broader structure-function knowledge base in the field, present capsid engineering opportunities, and enable the use of novel capsids with unique properties. Overall, CFT represents an unbiased method to evaluate novel AAV capsids, eliminating initial bias in tissue sampling and giving precedent for further investigation of tissues of interest.
 
Key Points:
Many naturally occurring AAV capsids remain under-studied and when describing biodistribution of novel vectors, a whole-body approach, such as CFT, in conjunction with traditional PCR-based biodistribution is preferable.

Microorganisms have always been integral to natural circular processes in the environment. They serve an important role in converting complex biological molecules into central building blocks that can be converted back into complex products. As a society, we now seek similar circularity in the semi-synthetic world that we’ve constructed through over a century of synthetic organic chemistry. Recent advances in genomics and synthetic biology have enabled exploitation of a wide array of microbes engineered to produce value-added products from biomass-derived and synthetically derived substrates. Our group has mainly focused on oleaginous yeast – that use a variety of low-value and waste feedstocks and can naturally accumulate a large amount of lipids. We have engineered multiple genetic tools to rapidly engineer Yarrowia lipolytica, and used these tools to elucidate cryptic metabolic pathways and to produce oleochemicals and secondary metabolites by taking advantage of naturally high flux pathways. We will also describe our work with emerging microbes that have more favorable properties for utilizing low-value and waste substrates, including lignin-derived aromatics and plastic waste.
 
Key Points:

• There is great potential for using non-conventional and non-model microbes to address sustainability issues in society.
• Our ability to rapidly develop genetic engineering tools enables us to take advantage of complex phenotypes non found in model microbes.
• We demonstrate the translation of tools development into improved processes for biochemical production.

Genetically modified cell models accelerate the development of safe and effective therapeutics, making them indispensable in the quest for new drugs. Through manipulation of cell line genetics, disease models can be faithfully replicated to screen and evaluate potential drug candidates. In particular, genetically engineered cell lines are essential for induced-proximity-based small-molecule drug discovery, enabling the expression of protein targets or disease-related proteins within cells. This facilitates the tracking of intracellular target engagement by protein ligands and monitoring changes in target abundance or modifications resulting from induced ternary complexes.

At Vicinitas Therapeutics, our vision centers around our proprietary Deubiquitinase Targeting Chimera (DUBTAC) platform, which aims to restore the levels of aberrantly degraded proteins that cause diseases, thereby conferring therapeutic benefits. To develop differentiated disease therapies based on our targeted protein stabilization platforms, cell lines are modified with lentivirus, transposon, and CRISPR to stably express specific proteins or variants involved in disease pathogenesis, such as tumor suppressors or mutated proteins causing genetic disorders. This allows us to study their effects, screen potential small-molecule drugs, and prevent their degradation.

Simultaneously, genetically engineered cell lines enable the utilization of multiple genetic and chemogenetic approaches to enforce proximity between target proteins and protein stabilization machinery. We employ a tagging system, iDimerize from Takara, which relies on FKBP-FRB-Rapamycin ternary, to stably express tagged protein targets and deubiquitinases in a selected tool cell line. By crossing the list of target proteins and deubiquitinases, we create a matrix that serves as a high-throughput assay system, capable of running hundreds of unique pairings concurrently. The resulting target stabilization and protein abundance change acts as a mimetic of induced proximity by future small-molecule DUBTACs. These proof-of-concept (PoC) studies play pivotal roles in early discoveries, aiding in target selection and validation.

Deubiquitinases reverse the effects of ubiquitination-dependent protein degradation via proteasome and lysosome, by cleaving the peptide or isopeptide bond between ubiquitin and its substrate protein. And a heterobifunctional molecule composed of target protein ligand, a linker, and a deubiquitinase recruiter ligand, facilitates the formation of an active ternary complex that leads to deubiquitination and restoration of target protein stability. Through genetic engineering of the cell model, we have developed cell-based assays that analyze the Mochenism-of-Action of DUBTAC and its chemogenetic mimetics. These assays utilize reporter cell lines and operate in heterogeneous systems that can be easily adapted into high-throughput workflows. They are able to quantify the intracellular target engagement or ligand binding, ubiquitination or deubiquitination, and ternary complex formation. These MoA studies provide a comprehensive understanding of DUBTAC function, guiding lead discovery and optimization of drug candidates.
By harnessing genetically engineered cell lines, the above approaches hold promise for drug discovery of diseases such as cancer and monogenic disorders, where the restoration of protein stability and function is crucial for therapeutic benefit. Vicinitas Therapeutics aims to overcome the challenge of targeting "undruggable" or intractable proteins, ultimately bringing about novel disease therapies that were previously inaccessible.

Learning Objectives:

• Vicinitas Therapeutics utilizes gene and cell engineering to develop differentiated disease therapies by stably expressing disease-related proteins and screening small-molecule drugs that prevent protein degradation.
• Genetically engineered cell lines facilitate induced-proximity-based small-molecule drug discovery by expressing disease-related proteins and tracking target engagement and ternary complex formation.

Chimeric antigen receptor (CAR)-reprogrammed immune cells offer exciting therapeutic potential for addressing oncology, autoimmune diseases, transplant medicine, and infections. However, current approved CAR-T therapies rely on personalized manufacturing using viral gene transfer, resulting in challenges related to CAR-signaling regulation and limited accessibility due to the expensive personalized manufacturing. To overcome these limitations, we present a novel approach utilizing non-viral CRISPR-Cas gene editing to redirect T cells or natural killer (NK) cells with CARs. By employing non-viral gene editing, we transfer truncated CAR-transgenes lacking the CD3ζ activation domain into the human CD3ζ (CD247) gene, thereby creating functional CAR fusion-genes. This strategy allows effective reprogramming of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. Notably, the engineered CD3ζ in-frame fusion gene in T cells eliminates TCR surface expression, substantially reducing the risk of graft-versus-host disease in allogeneic off-the-shelf applications. In a xenograft mouse model, optimized CD3ζ-CD19-CAR-T cells demonstrates remarkable leukemia control at low doses, outperforming CAR-T cells generated via TRAC gene editing. The CD3ζ-editing approach also enables reprogramming of NK cells without compromising their canonical NK cell functions in vitro. Collectively, CD3ζ gene editing represents a promising platform technology for the development of allogeneic off-the-shelf cell therapies utilizing redirected killer lymphocytes. 

Key Learning objectives:

• Non-viral gene editing with CRISPR-Cas and synthetic DNA templates allows efficient reprogramming of T cells for therapeutic applications.
• Integrating truncated CARs transgenes into the endogenous CD3ζ gene conveys potent CAR-dependent activation in various immune cells, such as T cells, regulatory T cells and natural killer cells.
• CD3ζ-edited CAR T cells are unable to express potentially allo-reactive T cell receptors and benefit from physiological CAR regulation.