Nanomaterials have already begun to play a pivotal role in overcoming the limitations of traditional drugs by enhancing drug delivery, improving bioavailability, overcoming biological barriers in the treatment and prevention of diseases. Spherical Nucleic Acids (SNAs) stand out as a versatile class of nanomaterials that have been utilized in applications spanning diagnostics and therapeutics. SNAs, comprised of a nanoparticle core densely functionalized with radially oriented oligonucleotides, offer unique advantages for nanomedicine. Their 3-dimensional structure renders SNAs with superior properties in comparison to linear nucleic acid molecules, including enhanced cellular uptake, resistance to nuclease degradation, and superior target affinity. These qualities make SNAs ideally suited for their application towards effective gene editing technologies. This presentation will cover two recent examples of employing SNAs in gene editing paradigms. First, an approach that enhances the therapeutic potential of small interfering RNAs (siRNAs) when formulated as SNAs is described. Until now, the gene silencing activity of SNAs had been hindered by endosomal entrapment. Here, a simple yet innovative strategy that employs calcium chloride (CaCl2) salting in SNA formulation results in enhanced cytosolic delivery and gene silencing activity by up to 20-fold across diverse cell lines, indicating a promising avenue for overcoming existing limitations. In a second, related story, SNAs are employed for the purposes of performing CRISPR/Cas9 systems for genome editing. In this work, Cas9 protein-cored SNAs, densely modified with DNA and preloaded with single-guide RNA, exhibit enhanced cellular uptake and stability, achieving notable genome editing efficiencies across multiple cell lines. The integration of GALA peptides and nuclear localization signals further enhances endosomal escape and nuclear localization. Taken together, this work underscores the importance of precise control over molecular interactions at the nanoscale and the potential of SNA-based nanotechnologies for gene editing applications. 

Learning Objectives:

• Spherical Nucleic Acids (SNAs), exhibit properties making them ideal constructs for nanomedicine and effective gene editing technologies. This talk describes routes of employing SNAs to markedly improve the cytosolic delivery of siRNAs and enhanced genome editing and delivery of CRISPR/Cas9 platforms.

CAR-T cell therapies are now the established treatments for patients with relapsed and/or refractory B cell lymphomas, B cell acute lymphoblastic leukemia, and multiple myeloma. Through deep depletion of targeted malignancy cells, CD 19-targeted and BCMA-target CAR-T treatment could lead to long-term remissions, in some cases curative. Inspired by the success of hematological cancers, CAR T cells are studied for diseases beyond oncology. A recent case study suggested deep depletion of B cells could alleviate the pathological progression and achieve sustained drug-free remission for autoimmune diseases such as systemic lupus erythematosus (SLE). Autoimmune diseases are “controlled” but incurable with the current treatment options due to their complex etiology. Cell therapies targeting autoreactive immune cells may offer a potentially curative approach in certain autoimmune diseases.

Key Learning Objectives:

• Review recent progress of cell therapies in autoimmune diseases
• Discuss the potential opportunities of CAR-T treatment in autoimmune disorders

Immunotherapy using chimeric antigen receptor (CAR) expressing NK cells engineered via viral vectors has shown exceptional efficacy against hematologic cancers in clinical trials. This method, however, is limited in cargo capacity and large-scale manufacturing for clinical use is cost-prohibitive to most academic centers. We have addressed these shortcomings by engineering CAR-NK cells using non-viral transposon engineering that can be paired with concurrent CRISPR/Cas9 editing. We believe that this approach avails opportunities that will allow researchers to address some of the more difficult solid tumor types.

Here, we developed a CAR-NK therapy with specific advantages to killing mesothelin-positive ovarian cancers which tend to form solid tumors, a new area for CAR NK cells. We started by testing 3 different mesothelin binders named SS1, 121, and M912. Our transposon vector also encodes for secretion of the cytokine IL15 for increased persistence and function of CAR-NK cells. The SS1, 121, and M912 CAR NK cells with soluble IL15 (Armored-NK) were tested in vitro and in vivo. No CAR variant dominated during the in vitro testing, so all three proceeded into a pilot in vivo study. In this experiment, NSG mice were engrafted with a mesothelin high tumor cell line, A1847, engineered to express Luciferase. A1847 cells were injected 14 days prior to the Armored CAR NK injection. Both tumor cells and Armored CAR NK cells were delivered via intraperitoneal (IP) injection.

At week 6, control mice, 121 treated, and M912 treated mice had tumors beginning to grow exponentially while the SS1 CAR controlled tumor growth. This trend continued for 3 more weeks as the SS1-treated mice did not see significant tumor growth while all other mice had to be euthanized due to tumor burden. SS1 mice survived long term with the only detectable tumor being a mass that grew at the injection site, outside of the IP space. With these results, we plan to test additional edits and modifications to SS1 cytokine-armored CAR-NK cells before proceeding with a phase 1 clinical trial. We believe these edits will enable cytokine-armored CAR-NK cells to treat solid tumors.

Key Learning Objectives:
Natural killer (NK) cells offer an off-the-shelf therapy for cancer immunotherapy
NK cells can be efficiently engineered using non-viral DNA transposons delivered as GenCircle plasmids
Transposon-engineered, cytokine-armored CAR-NK cells are effective against ovarian cancer in vitro and in vivo

Bacteria defend themselves from viral infection using diverse immune systems, many of which sense and target foreign nucleic acids. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this immune strategy by instead leveraging DNA synthesis, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems execute an unprecedented immunity mechanism that involves de novo gene synthesis via rolling circle reverse transcription of a non-coding RNA (ncRNA). Unbiased profiling of RT-associated RNA and DNA ligands in DRT2-expressing cells revealed that reverse transcription generates concatenated cDNA repeats through programmed template jumping on the ncRNA. The presence of phage then triggers second-strand cDNA synthesis, leading to the production of long double-stranded DNA. Remarkably, this DNA product is efficiently transcribed, generating messenger RNAs that encode a stop codon-less, never-ending ORF (neo) whose translation causes potent growth arrest. Phylogenetic analyses and screening of diverse DRT2 homologs further revealed broad conservation of rolling circle reverse transcription and Neo protein function. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.

Learning Objectives: 

• Bacterial antiviral defense systems have served as a treasure trove for the discovery of genome engineering tools, such as restriction enzymes and CRISPR-Cas nucleases.
• Most bacterial defense systems target and cleave DNA, but defense-associated reverse transcriptases synthesize DNA during an antiviral response.
• DRT2 systems exhibit an unprecedented mechanism of rolling circle reverse transcription, leading to the amplification of concatenated cDNA products.

Tome Biosciences is leading the field of programmable genomic integration (PGI). Building on founding technologies obtained from MIT and Replace Therapeutics, Tome has developed and refined PGI resulting in stable integration of multiple kilobases of DNA at user-defined locations. Integrase-mediated PGI (I-PGI) uses a Cas9 nickase with a writing enzyme (reverse transcriptase or ligase) to write an integrase recognition site (‘beacon’) at a user-defined location in the genome. Beacon placement (BP) is followed by integrase-mediated integration of donor DNA. I-PGI enables therapeutic correction of genetic diseases through the insertion of functional genes at the endogenous location without dependency on a double-strand break (DSB) and is not dependent on homology-directed repair (HDR). This technology is agnostic to the size of the DNA template and, due to the orthogonal nature of the integrase: beacon system and lack of DSBs, this technology can be used to generate complex cell therapies via multiplex large gene insertion.

This presentation will focus on an overview of PGI and highlight the progress Tome has made in developing this technology into medicine for patients in need. We will present data showing efficient PGI in primary human cells (iPSCs, Primary Human Hepatocytes (PHH)), as well as our current progress in translating PGI into rodent and non-human primates (NHP).

Tome is developing this technology to treat a wide variety of both rare and common diseases. For in vivo gene therapy applications, PGI will enable the integration of a healthy copy of a gene at the endogenous location, meaning that a single product will work for the majority of the patient population, regardless of the specific mutation. For cell therapy applications, PGI enables the development of highly edited cell therapies, opening the door to a new generation of medicine specifically designed for common diseases such as autoimmunity.

Learning Objectives:
PGI is a new technology that combines site specificity of integrases with Cas9-based genome editing to allow large gene insertion/replacement at user-defined locations.