GenScript 4^rd Annual
Gene & Cell Engineering Virtual Summit

Keynote Presentation

Exploring the Development & Optimization of mRNA Delivery Systems to Advance mRNA-based medicines

Dr. Jason Zhang is a Co-Founder and CEO of Zipcode Bio, a company dedicated to developing mRNA vaccines and therapeutics. In his talk, Dr. Zhang discussed different strategies leveraged by the Zipcode Bio team to improve the expression and efficacy of mRNA-based medicines. He also introduced their innovative delivery platform to achieve tissue-specific targeting of mRNA therapies.

mRNA-based medicines have a broad range of applications, including vaccines, protein replacement, ex vivo and in vivo cell therapies (e.g., CAR-Ts), genome editing, and therapeutic antibody production. mRNA vaccines are now developed through a well-established platform enabling high efficacy, as exemplified by those developed against SARS-CoV-2 and RSV immunogens. Improving further is always possible, especially in developing effective mRNA therapeutics which would require efficient and targeted expression. Additionally, for mRNA-based therapeutics, Dr. Zhang emphasized that demonstrating the advantages of mRNA as a modality of choice is imperative. For instance, mRNA-based therapeutics should demonstrate benefits in pharmacokinetics and pharmacodynamics (PK/PD) and efficacy in comparison to alternative modalities such as protein-based therapeutics (e.g., antibodies). One of the mRNA therapeutics Dr. Zhang has worked to optimize encodes an antibody for the treatment of Clostridium difficile infections.

At Zipcode Bio, the team leveraged design–build–test–learn iterative cycles to develop novel 5’ and 3’ UTR sequences that achieve enhanced stability and translation efficiency. For delivery, LNPs are currently the main vehicle for the delivery of mRNA. Zipcode Bio has optimized its LNP formulation to achieve lower immunogenicity, which is critical for their use in delivering mRNA therapeutics. Beyond optimizing traditional LNPs, Zipcode Bio has developed an innovative technology to ensure tissue-targeted mRNA therapeutic delivery. Their technology, DNP/SHARP, enables the production of single-component nanoparticles to package mRNA, achieving better efficacy, safety, and stability than traditional four-component LNPs. Significantly, optimized designs of their novel single-component chemical structure have enabled the specific targeting of several tissues, including the spleen and lung.

Talk 1

De novo gene synthesis by an antiviral reverse transcriptase

Dr. Sternberg shared work from his group at Columbia University focused on understanding a newly discovered antiviral immune system in bacteria. His lab has a long record of work uncovering mechanisms of bacterial antiviral immune systems, such as CRISPR/Cas9 and mobile genetic elements or Transposons. More recently, the lab has focused on antiviral immune systems using repurposed enzymes from transposons. An overarching goal in the lab is to leverage their findings to develop new tools for genome engineering.

Immune systems that target viral DNA for degradation, such as restriction enzymes and CRISPR/Cas9, figure prominently across bacteria. However, in his talk, Dr. Sternberg highlighted the great number of antiviral immune systems that remain unexplored and uncharacterized. His recent work, led by Stephen Tang, an MD/PhD student in the lab, focuses on one such novel system, defense-associated reverse transcriptase or DRT. Intriguingly, rather than cutting and degrading DNA, this new system relies on DNA synthesis as an antiviral mechanism.

To fully understand the inner workings of the DRT system, the team had to address many unknown features through careful and complex experimentation, including identifying the RNA substrate, the resulting cDNA product, and the antiviral mechanism at play. What emerged from their studies is a complex sequence of DNA, RNA, and protein synthesis, all starting from a noncoding RNA. From their findings, they have proposed growth inhibition as the ultimate anti-viral mechanism at play, which is mediated by the resulting protein product.

Talk 2

Cytokine Armored CAR-NK Cell Therapy for Advanced Ovarian Cancer

Dr. Moriarity's team is developing cell therapies based on modified NK cells. His talk focused on current work towards a Phase I clinical trial with ovarian cancer patients using a highly engineered CAR-NK cell therapy. The selection of NK cells for engineering cell therapies relates to several advantages, including their inherent tumor-killing ability, capacity to engage in antibody-dependent cellular cytotoxicity (ADCC), and capability for enhanced cancer cell targeting via CARs. Engineering strategies for NK cells have advanced significantly over the years, motivated by their lower risk of adverse reactions, such as graft versus host disease, cytokine release syndrome, and neurotoxicity when used as allogeneic therapies. Additionally, advanced strategies for their expansion are well established. Therefore, engineered NK cells, as opposed to T cells, provide greater opportunities for ready-made therapies to benefit a larger population.

NK cells may be obtained from different sources, including peripheral blood, differentiated from pluripotent stem cells, or cell lines. Dr. Moriarity's efforts have been focused on engineering NK cells directly selected from peripheral blood mononuclear lymphocytes (PBMCs), which are further expanded using feeder cultures. PBMC-derived NK cells are readily available from donors, have a true NK phenotype, and have proven safety records based on previous clinical trials.

To target ovarian cancer cells, the NK cells have been engineered to express an anti-Mesothelin CAR. Additionally, to promote the function and persistence of their CAR-NK cells, Dr. Moriarity's team has further modified the cells to express IL-15. Lastly, the cells have also been modified to express the cell surface receptor RQR8, which serves as a marker for selection and provides a mechanism to eliminate the cells if needed due to toxicities. Significantly, Moriarity's team has opted to engineer NK cells through a fully non-viral approach, leveraging transposons, specifically the TC Buster transposon system, which they have further improved to increase the transposase’s activity. By leveraging this system and a minimal backbone plasmid for payload delivery via GenScript’s GenCircle, the team has achieved over 50% efficiency in generating highly engineered IL-15 armored CAR-NK cells. A definite advantage of the transposon system is the cost associated with the GMP reagents required, particularly Dr. Moriarity emphasized that “GMP transposon can be produced in 4-5 months for <$400,000,” supporting the production of upwards of 1,000 doses of therapeutic cells.

To date, Moriarity’s group has mostly finalized in vitro and in vivo IND application-enabling studies using the highly engineered IL-15 armored CAR-NK cells. Their in vivo studies have demonstrated that their cell product is efficacious and has a long persistence, eradicating tumors and extending life span in animal models. Therefore, with their already established GMP-compliant manufacturing process for generating this cell therapy, they are well-positioned to finalize their IND application by the end of the year and commence Phase I clinical trials in 2025.

Talk 3

Programmable Genomic integration (PGI): a technology that enables the integration of large DNA sequences at specific genomic locations

At Tome Biosciences, Dr. Sandeep Kumar’s team works to develop new strategies for efficient and specific genome editing through programmable genomic integration (PGI). This technology allows the incorporation of large-size payloads at user-defined locations in the genome. Directional incorporation of genes or large gene segments downstream of their corresponding regulatory sequences, such as promoters and enhancer sequences, enables retaining physiological gene expression. Dr. Kumar’s team has been working to improve the efficiency of integration to achieve in primary clinically relevant cells the high levels of integration achieved with cell lines, such as HEK cells (80-90%). While the technology has the potential to achieve integration of any cargo size, Dr. Kumar pointed out that current DNA-size delivery options certainly restrict this capacity. Lastly, Tome’s technology does not involve DNA double-strand break and is amenable to multiplexing.

Tome pioneering technologies leverage Cas9 nickase and rely on several tools, including those supporting Ligase-mediated or Integrase-mediated PGI, for genomic integration of smaller (e.g., ~100 bps) and larger sequences (e.g., over 1,000 bps), respectively.

Dr. Kumar shared that I-PGI works as a two-step process. First, a beacon (~40 bps) is placed specifically within the genome by the combined use of Cas9 nickase and a writing enzyme. In the second step, the beacon serves as an integrase-recognition sequence, enabling Tome’s proprietary integrase to insert the payload specifically.

These technologies are leveraged by Tome to develop cell therapies by multiplex cell engineering and for in vivo applications. For example, I-PGI has been implemented by the Tome team for the multiplex engineering of iPSCs that can be further differentiated into different cell types of choice and, therefore, can potentially serve as therapies for various disease indications. Additionally, for in vivo application, I-PGI technology enables Tome to restore gene function by integration of normal gene sequences directly downstream of corresponding endogenous promoters.

Talk 4

The Power of Automation: How Robotics Can Dramatically Reduce Cell Therapy Manufacturing Costs and Scale Patient Access

The stringent manufacturing workflows necessary for developing cell therapies impose great challenges to those in the industry who must meet the high demands of personalized medicines. Automation of these workflows is increasingly needed to expedite production while adhering to manufacturing standards.

In his talk, Dr. Fred Parietti introduced Multiply Labs' mission: to partner with industries in the cell therapy space to facilitate the production of medicines at an industrial scale via automated systems. Multiply Labs has developed its automated technology to serve the broad cell-engineered therapy space. By concentrating on the common requirements and equipment generally leveraged in cell therapy development, Multiply Labs provides all-inclusive technologies to streamline and simplify these workflows.

Their technology is designed to be compatible with a broad range of instruments commonly used by cell therapy manufacturing industries globally. To achieve this goal, Multiply Labs adapts and trains its robotic technologies to achieve compatibility with industry-leading GMP instruments and processes. Dr. Parietti shared that a core tenet of the Multiply Labs’ approach is that “we don’t force cell therapy developers to change their process; we adapt to it.” Therefore, in partnering with manufacturers, Multiply Labs strives to introduce automation that replicates established processes, including the use of specific instruments, reagents, and consumables, as well as any necessary manual steps, such as cell resuspensions and mixing tasks.

The modular nature of their robotic technology provides flexibility to satisfy the needs of manufacturers requiring to automate only specific steps in their established workflows. Moreover, it enables manufacturers to introduce new automation as needed based on evolving processes and new regulatory requirements. To date, Multiply Labs has automated several instruments commonly used in cell therapy manufacturing for various functions, including sterilization, washing, enrichment/isolation (GenScript’s CytoSinct), gene editing/expansion, and harvesting.

Talk 5

Synthetic biology approaches to improve cell therapy function

GeneFab aims to leverage its expertise, technological know-how, and platforms for high-throughput synthetic biology, informatics, and AI to support gene and cell therapy development. Dr. Hung’s talk focused on various GeneFab synthetic biology solutions designed to help overcome critical challenges in cell therapy development, including achieving precise targeting, maintaining therapeutic function, and accessing therapeutic windows.

As a principal scientist in the synthetic biology group at GeneFab, Dr. Hung’s team helps provide end-to-end solutions for customers developing next-generation therapies, from initial data analysis, library generation, design of custom functional assays, and high-throughput screening to candidate optimization and validation.

Dr. Hung shared how GenFab’s synthetic biology task force has supported various projects for leading Cell and Gene Therapy Companies, including Senti Bio, BlueRock Therapeutics, and Spark Therapeutics. For example, the team supported Senti Bio by developing custom gene circuits for boosting CAR-NK safety and efficacy and Sparks Therapeutics in custom promoter discovery for their AAV gene therapy platform.

Talk 6

Advancing Gene Editing Technologies with Spherical Nucleic Acids

Modulation of gene expression through various approaches provides opportunities for various disease states, including rare diseases, cancer, and more. Among various gene modulating strategies, antisense oligonucleotides (ASO) and small interfering RNA (siRNA) are very specific and silence gene expression via different mechanisms. ASOs, consisting of single-stranded DNA or RNA, hybridize with mRNA molecules resulting in their degradation and inhibiting translation. In siRNA, a double-stranded RNA is used, which is processed by the cellular machinery into smaller fragments that ultimately target specific mRNAs for degradation. However, as pointed out by Dr. Sergej Kudruk, while these approaches are highly specific, they also carry various disadvantages related to their molecular properties, which make them susceptible to degradation and fast clearance. Moreover, ASOs and siRNAs as charged molecules have limited capacity to traverse cell membranes.

Dr. Kudruk shared his work developing tridimensional nucleic acid structures or Spherical Nucleic acids (SNAs) to improve the performance of various gene editing tools. SNAs consist of a core (e.g., lipid, PLGA, or protein) surrounded by a nucleic acid shell. This modular platform offers various benefits, including increased stability, cellular uptake, and reduced immunogenicity. Moreover, nanoparticle composition can be optimized for access to specific cellular compartments, including cytosol and nuclei, among others.

SNAs have been shown in various model systems, such as retinal disease (e.g., VEGF-siRNA-SNA), to efficiently deliver nucleic acids with therapeutic benefit. Of significance, as emphasized by Dr. Kudruk, SNAs (i.e., miRNA) can traverse the blood-brain barrier, providing efficient delivery of gene-modulating molecules to target glioblastomas, reducing tumor size and extending survival in mice.

SNAs are not without challenge, as limitations in endosomal escape can prevent them from reaching desired cellular compartments and their therapeutic effects. Therefore, Dr. Kudruk shared his work on the optimization of SNAs based on a PLGA core to promote access to the cystosol.

Talk 7

Autoimmune Diseases, the Arising Opportunities for Cell Therapies

In her talk, Dr. Dong Geng first highlighted the success of CAR-T cell therapies in hematological malignancies, such as B-cell leukemia, lymphoma, and multiple myelomas, where high efficacy and long-term disease remission has been achieved. To date, six CAR-T cells targeting B-cell antigens CD19 or BCMA are FDA-approved for cancer therapy.

Beyond cancer, autoimmune diseases are emerging as potential indications for the implementation of CAR-T cell therapies. Currently, autoimmune disease therapies include anti-inflammatory drugs and immunosuppression, which do not provide a curative solution.

B cells play a central role in autoimmune diseases, such as multiple sclerosis, lupus, rheumatoid arthritis, and more. Acting through different mechanisms, including antigen presentation, inflammation, and auto-antibody production, B cells damage healthy tissue and organs. Because of this central role, antibody drugs, such as rituximab, targeting CD19, have shown benefits as therapies for autoimmune diseases. However, Dr. Geng pointed out that responses to this antibody-drug are not durable due to incomplete B cell clearance. As opposed to antibody drugs, CAR-T cells targeting B-cell antigens may more effectively lead to prolonged B-cell depletion.

To date, clinical studies using CAR-T cells (e.g., anti-CD19) as therapies for autoimmune diseases indicate that deep B-cell depletion is achieved and associated with reduced autoantibody production and remission. Significantly, following CAR-T cell treatment, B-cells are reconstituted within ~4 months in lupus patients, but the cells are phenotypically different, being mostly naïve. Therefore, the reduced number of antibody-producing, memory B cells and, consequently, autoantibodies after CAR-T cell therapy suggests that this approach may be effective at re-setting autoimmunity. Additional clinical studies suggest similar outcomes with prolonged remissions of ~1 year or longer, enabling patients to discontinue immunosuppressive therapies for up to 2 years. Similarly, in clinical studies with patients with Myasthenia Gravis, treatment with anti-BCMA CAR-T cells was effective in reducing disease severity.

Driven by promising findings, the number of clinical trials evaluating the use of CAR-T cells in autoimmune disease patients has increased significantly, from fewer than 10 in 2021 to 40 studies in 2024. Despite this increase, studies so far are relatively small, and therefore more data is required to fully understand the full benefits and long-term effects of CAR-T cells in this heterogeneous patient population.