• Dysregulated RNA processing is emerging as a new hallmark of aging. 
• Correcting aberrant regulation of splicing regulators is capable of rescuing multiple features of cellular senescence in human primary cells of multiple cell types.
• Restoration of splicing homeostasis may therefore underpin promising novel anti-degenerative therapies in the future.

Production of pDNA suffers from the low productivity of microbial fermentation and the purification process remains challenging. This presentation describes an end-to-end platform where each of the steps will be explored, along with strategies to optimize and streamline the purification workflow. In addition, considerations for an mRNA template process are discussed.

The development and approval of mRNA-based vaccines for COVID-19 revealed the potential of this platform for both preventative and therapeutic purposes. A standard set of analytical methods to assess mRNA quality would support product developers, manufacturers, regulators, and control laboratories worldwide. In collaboration with vaccine experts, USP has developed draft guidelines with analytical procedures for quality assessment of mRNA products. Results of this work and stakeholder input will be shared.

mRNA-lipid nanoparticle vaccines provide many advantages inclusive of antigen specificity and rapid vaccine development. Characterization of this platform is relatively novel, yet requisite for vaccine production and licensing. Therefore, a cell-based assay was developed to quantify transgene protein expression efficiency and product potency in this platform. Appropriate assay factors were characterized, inclusive of monolayer morphology and mRNA-cassette protein expression kinetics, and effects of lipid nanoparticle properties were evaluated.

siRNAs are a new class of therapeutic moieties that harness endogenous RNAi process to enable specific and sustained silencing of mRNAs, thus reducing the protein synthesis of a disease target. RNAi allows rapid drug discovery due to the ease of sequence-based design and the ability to target disease genes previously considered as "undraggable." We chemically engineer siRNAs for various therapeutic applications, and here we present the preclinical development of fully chemically-modified siRNAs targeting IFN-gamma pathway to establish a path toward the treatment of autoimmune skin diseases.

In the present study, three-dimensional steady-state CFD simulations for varying sparge rates and impeller agitation rates are used to develop a reduced-order model of bioreactor performance.  This model is coupled to a zero-dimension transient model representing various cellular metabolic processes.  A coupled system-level control model is then developed using Ansys TwinBuilder and exported as a digital twin.  

We describe here a well-established platform process for LV production based on transient transfection of serum-free cells grown in suspension. Both upstream and downstream processes are highly optimized to achieve optimal vector yields and significant decrease in the impurities (host cell protein/DNA, plasmid DNA). The compatibility of this platform process has been evaluated with multiple CAR/TCR genes while the robustness is demonstrated in reproducible runs at pilot scale.

Umoja aims to transform cancer care by creating an off-the-shelf, direct injection lentiviral vector (LVV) drug product for in vivo CAR T cell generation and expansion. We will present our approach to reproducible and scalable manufacturing of high-quality LVV, with special focus on expediting process development with risk-based quality documentation and impurity clearance at key process unit operations to meet ambitious final specifications.

To streamline lentiviral vector (LV) manufacturing, we propose to use packaging cell lines in suspension cultures. The cells were designed using molecular switches to control the production of LV cytotoxic proteins. They can be used to generate LV through a one-plasmid transfection corresponding to the gene of interest, or to generate stable producer cell lines. Given the labile nature of the vector, we are evaluating the advantages of producing in perfusion mode, and the possibility of purifying in a semi-continuous way.

The traditional tools used for protein-based therapeutics often come short in case of viruses. This is particularly the case when the virus is to be used for oncolytic purposes rather than as a vaccine wherein the administered dosages are several-fold lower. Another major gap with viral vectors is in our knowledge of the biology and morphology of the therapeutic. The work presented will focus on our efforts in development of novel chromatographic purification techniques complimented with extensive characterization of our virus using a suite of analytics.

There are a number of challenges drug developers face on the journey to the clinic, including high costs and competitive timelines. FDB has solutions that help our partners navigate the challenges of development and material supply for clinical trials as well as propel their life changing medicines to commercialization. Our solutions include a flexible AAV platform and a wealth of regulatory experience taking medicines to the market for our partners.

Lipid nanoparticles (LNPs) have proven their value for delivery of RNA medicines in the clinic. We argue that some critical needs should be addressed to facilitate and accelerate the next-generation of LNPs for nucleic acid delivery, for the benefit of patients. These needs include i) automated platforms for combinatorial synthesis linked to high-throughput in vitro and in vivo screening systems; ii) mRNA and/or LNP reference materials, and iii) robust test methods and protocols.

Recently, the development of gene editing technologies (CRISPR-Cas,TALENs, zinc finger nucleases (ZFNs)) have opened new opportunities to precisely edit the genome, target disease causing mutations, and potentially enable one-time cures of genetic diseases. However, these therapeutics must overcome numerous obstacles to be successful, including rapid in vivo degradation, poor uptake into target cells, required nuclear entry, and potential in vivo toxicity in healthy cells and tissues. In this talk, I will discuss our efforts towards the development of lipid and polymer-based nanoparticles that enable the delivery of nucleic acid therapeutics to target cells and tissues in vivo. Furthermore, I will describe new therapeutic strategies utilizing these nanoparticles to (i) reprogram immune cells for cancer immunotherapy applications, (ii) in utero mRNA delivery for treating disease before birth.

We are developing a single-shot, room temperature stable mRNA vaccine by double nanoencapsulating the mRNA construct in phospholipid nanosomes and biodegradable polymer nanospheres. We are using continuous flow, solvent-free processes that minimize loss of potency, preserve antigenicity of the nanoencapsulated mRNA, and eliminate residual organic solvents. The impact of this development would be significant to the US and worldwide vaccination for current and future coronavirus pandemics and other infectious diseases.