Delivery Of Therapeutic Proteins Research Articles

Hypoxia-Responsive Hydrogen-Bonded Organic Framework-Mediated Protein Delivery for Cancer Therapy.

The efficient delivery of therapeutic proteins to tumor sites is a promising cancer treatment modality. Hydrogen-bonded organic frameworks (HOFs) have been successfully used for the protective encapsulation of proteins; however, easy precipitation and lack of controlled release of existing HOFs limit their further application for protein delivery in vivo. In this study, a hypoxia-responsive HOF, self-assembled from azobenzenedicarboxylate/polyethylene glycol-conjugated azobenzenedicarboxylate and tetrakis(4-amidiniumphenyl)methane through charge-assisted hydrogen-bonding, is developed for systemic protein delivery to tumor cells. The newly generated HOF platform efficiently encapsulates representative cytochrome C, demonstrating good dispersibility under physiological conditions. Moreover, it can respond to overexpressed reductases in the cytoplasm under hypoxic conditions, inducing fast intracellular protein release to exert therapeutic effects. The strategy presented herein can be applied to other therapeutic proteins and can be expanded to encompass more intrinsic tumor microenvironment stimuli. This offers a novel avenue for utilizing HOFs in protein-based cancer therapy. This article is protected by copyright. All rights reserved.

Polyphenol-Assisted Biomineralization of Metal-Organic Framework Nanoparticles for Precision Delivery of Therapeutic Proteins to Cancer Cells.

The delivery of proteins into the cytosol holds great promise for cell signaling manipulation and the development of precision medicine. However, this potency is challenged by achieving targeted and controlled delivery, specifically within diseased cells. In this study, we introduce a versatile and effective method for the precision delivery of therapeutic proteins to cancer cells by designing polyphenol-assisted biomineralization of zeolite imidazole framework-8 (ZIF-8). We demonstrate that by leveraging the strong noncovalent binding affinity of epigallocatechin gallate (EGCG) with both proteins and ZIF-8, our approach significantly enhances the biomineralization of ZIF-8, which in turn improves the efficiency of protein encapsulation and intracellular delivery. Moreover, the incorporation of EGCG within ZIF-8 enables controlled degradation of the nanoparticles and the selective release of the encapsulated proteins in cancer cells. This selective release is triggered by the oxidation of EGCG in response to the high levels of reactive oxygen species (ROS) found within cancer cells that destabilize the EGCG/ZIF-8 nanoparticles. We have further demonstrated the ability of EGCG/ZIF-8 to deliver a wide range of proteins into cancer cells, including bacterial virulence protein, to rewire cell signaling and prohibit tumor cell growth in a mouse xenograft model. Our strategy and findings underscore the potential of designing the EGCG/ZIF-8 interface for specific and controlled protein delivery for targeted cancer therapy.

Engineered extracellular vesicles enable high-efficient delivery of intracellular therapeutic proteins.

Developing an intracellular delivery system is of key importance in the expansion of protein-based therapeutics acting on cytosolic or nuclear targets. Recently, extracellular vesicles (EVs) have been exploited as next-generation delivery modalities due to their natural role in intercellular communication and biocompatibility. However, fusion of protein of interest to a scaffold represents a widely-used strategy for cargo enrichment in EVs, which could compromise t the stability and functionality of cargo. Herein, we report intracellular delivery via EV-based approach (IDEA) that efficiently packages and delivers native proteins both in vitro and in vivo without the use of a scaffold. As a proof-of-concept, we applied the IDEA to deliver cyclic GMP-AMP synthase (cGAS), an innate immune sensor. The results showed that cGAS-carrying EVs activated interferon signaling and elicited enhanced antitumor immunity in multiple syngeneic tumor models. Combining cGAS EVs with immune checkpoint inhibition further synergistically boosted antitumor efficacy in vivo. Mechanistically, scRNA-seq demonstrated that cGAS EVs mediated significant remodelling of intratumoral microenvironment, revealing a pivotal role of infiltrating neutrophils in the antitumor immune milieu. Collectively, IDEA, as a universal and facile strategy, can be applied to expand and advance the development of protein-based therapeutics.

Abstract 3254: Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines

Abstract mRNA-based therapeutics have shown clinical efficacy, evidenced by the success of the mRNA vaccines for SARS-CoV-2. Personalized mRNA vaccines containing patient-derived tumor mutations are now entering the clinic and have shown promise. Delivery of mRNA-encoded therapeutic proteins (e.g., antibodies) is also under development. mRNA drug substance is typically generated by in vitro transcription (IVT). One byproduct and contaminant of IVT is the formation of double-stranded RNA (dsRNA). dsRNA is highly immunogenic, can induce inflammation and cell death, and inhibit protein translation, including the protein encoded by the therapeutic mRNA. As such, dsRNA must be measured for all IVT products to ensure safety and efficacy. However, existing dot bot and ELISA technology to detect dsRNA is neither sensitive nor quantitative. Herein, we describe the development of novel assays for sensitive and specific quantitation of dsRNA in IVT products and following encapsulation of IVT products in lipid nanoparticles (LNPs). First, NanoBiT® split luciferase technology was applied to detect dsRNA in IVT products. dsRNA binding domains were genetically attached to either SmBiT or LgBiT. In the presence of dsRNA, the SmBiT and LgBiT constructs bind via the dsRNA binding domains, resulting in dimerization and the complementation of functional NanoBiT® luciferase. NanoBiT® then generates light which is proportional to the amount of dsRNA in the sample. The assay detects dsRNA greater than or equal to 30 base pairs and is independent of the size and sequence of the dsRNA contaminant. The assay does not cross-react with single-stranded RNA or DNA and is highly sensitive, with a limit of detection less than 100 pg/ml. As a complementary tool to the biochemical assay, we developed a cell-based bioassay to detect dsRNA encapsulated in LNPs. dsRNA/LNPs are taken up by endocytosis where Toll-like receptor 3 (TLR3) detects dsRNA. Activation of TLR3 initiates a signaling pathway that is detected by a TLR3 pathway-specific promoter driving luciferase expression. This pathway likely represents the most physiologically-relevant readout of dsRNA bioactivity. Both assays are developed in “add-mix-read” format with no wash steps or media transfer, and exhibit robust, sensitive, and repeatable performance for specific detection of dsRNA in mixed solutions. Citation Format: Rich Moravec, Jun Wang, Jim Hartnett, Mei Cong, Jamison Grailer. Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3254.

The encapsulation and controlled release of proteins from “meltable” chemically cross‐linked hydrogels

AbstractChemically crosslinked hydrogels can be used for the controlled delivery of therapeutic proteins, however, cargo loading can be challenging. A common approach involves performing the hydrogel crosslinking step in the presence of the cargo proteins, however, this can require the utilization of selective chemistries to ensure the cargos do not chemically react with the forming hydrogel network. We demonstrate that chemically crosslinked hydrogels based upon the protein polymer Capsular antigen fragment 1 (Caf1) can be used to encapsulate proteins within a chemically crosslinked hydrogel network. This encapsulation exploits the “meltable” feature of Caf1 hydrogels, where upon heating the Caf1 protein unfolds with concomitant depolymerization of the hydrogel network. Protein cargos can be loaded into the “molten” form of the hydrogel which cools with protein refolding and concomitant reformation of the hydrogel network to entrap the cargos, ensuring a more homogenous dispersion of the cargo. Analysis of release profiles indicated lower burst release in comparison to release from identical hydrogels loaded by an absorption method. This work is significant because it suggest that the “meltable” feature of Caf1 hydrogels may present an improved method of loading protein cargos into crosslinked hydrogel networks whilst avoiding the need for crosslinking chemistries.

Intracellular Delivery of Therapeutic Protein via Ultrathin Layered Double Hydroxide Nanosheets.

The therapeutic application of biofunctional proteins relies on their intracellular delivery, which is hindered by poor cellular uptake and transport from endosomes to cytoplasm. Herein, we constructed a two-dimensional (2D) ultrathin layered double hydroxide (LDH) nanosheet for the intracellular delivery of a cell-impermeable protein, gelonin, towards efficient and specific cancer treatment. The LDH nanosheet was synthesized via a facile method without using exfoliation agents and showed a high loading capacity of proteins (up to 182%). Using 2D and 3D 4T1 breast cancer cell models, LDH-gelonin demonstrated significantly higher cellular uptake efficiency, favorable endosome escape ability, and deep tumor penetration performance, leading to a higher anticancer efficiency, in comparison to free gelonin. This work provides a promising strategy and a generalized nanoplatform to efficiently deliver biofunctional proteins to unlock their therapeutic potential for cancer treatment.

Insulin Delivery to the Brain via the Nasal Route: Unraveling the Potential for Alzheimer's Disease Therapy.

This comprehensive review delves into the potential of intranasal insulin delivery for managing Alzheimer's Disease (AD) while exploring the connection between AD and diabetes mellitus (DM). Both conditions share features of insulin signalling dysregulation and oxidative stress that accelerate inflammatory response. Given the physiological barriers to brain drug delivery, including the blood-brain barrier, intranasal administration emerges as a non-invasive alternative. Notably, intranasal insulin has shown neuroprotective effects, impacting Aβ clearance, tau phosphorylation, and synaptic plasticity. In preclinical studies and clinical trials, intranasally administered insulin achieved rapid and extensive distribution throughout the brain, with optimal formulations exhibiting minimal systemic circulation. The detailed mechanism of insulin transport through the nose-to-brain pathway is elucidated in the review, emphasizing the role of olfactory and trigeminal nerves. Despite promising prospects, challenges in delivering protein drugs from the nasal cavity to the brain remain, including enzymes, tight junctions, mucociliary clearance, and precise drug deposition, which hinder its translation to clinical settings. The review encompasses a discussion of the strategies to enhance the intranasal delivery of therapeutic proteins, such as tight junction modulators, cell-penetrating peptides, and nano-drug carrier systems. Moreover, successful translation of nose-to-brain drug delivery necessitates a holistic understanding of drug transport mechanisms, brain anatomy, and nasal formulation optimization. To date, no intranasal insulin formulation has received regulatory approval for AD treatment. Future research should address challenges related to drug absorption, nasal deposition, and the long-term effects of intranasal insulin. In this context, the evaluation of administration devices for nose-to-brain drug delivery becomes crucial in ensuring precise drug deposition patterns and enhancing bioavailability.

Polymersomes for Therapeutic Protein and Peptide Delivery: Towards Better Loading Properties.

Therapeutics based on proteins and peptides have profoundly transformed the landscape of treatment for diseases, from diabetes mellitus to cancers, yet the short half-life and low bioavailability of therapeutic proteins and peptides hinder their wide applications. To break through this bottleneck, biomolecules-loaded polymersomes with strong adjustability and versatility have attracted more and more attentions recently. Loading proteins or peptides into polymersomes is the first but extremely important step towards developing high-quality formulation products. However, increasing protein and peptide loading content is quite challenging due to the inherent nature of self-assembled vesicle formation mechanism and physiochemical characteristics of biomacromolecules. This review highlights the potential of polymersomes as the next-generation therapeutic proteins and peptides carrier and emphatically introduces novel approaches and recent progress to achieve satisfactory encapsulation capability of polymersomes for proteins and peptides. On the one hand, with the help of intermolecular interactions, such as electrostatic, lipid-protein, and hydrophobic interactions, the drug loading could be significantly improved. On the other hand, loading improvement could be attained through innovation of preparation methods, ranging from modified traditional film hydration techniques to the novel phase-guided assembly method.

Organic clay-based fast dissolving microneedles for efficient transdermal delivery of therapeutic proteins

Organic clay-based fast dissolving microneedles for efficient transdermal delivery of therapeutic proteins

Circumventing the Gastrointestinal Barrier for Oral Delivery of Therapeutic Proteins and Peptides (PPTs): Current Trends and Future Trajectories.

Therapeutic proteins and peptides (PPTs) have become one of the most important biological molecules for the management of many common and complex diseases due to their high specificity and high bioactivity. However, these biomolecules are mainly given by the hypodermic injection, which often leads to poor patient compliance due to the invasive nature of this route of administration. The oral route has been considered the most convenient and patient-friendly route for drug delivery relative to hypodermic injection. Despite the ease and simplicity conferred by oral administration, this drug delivery route suffers rapid peptide degradation in gastric fluid and low intestinal uptake. In order to circumvent these issues, several strategies, such as enzyme inhibitors, permeation enhancers, chemical modification, mucoadhesive and stimuli-responsive polymers, and specialised particulate formulation have been developed. Such strategies are designed with the aim of protecting proteins and peptides from the harsh gastrointestinal environment as well as providing a strategy to enhance the uptake of the therapeutic across the gastrointestinal tract. This review aims to provide an overview of the current development in enteral drug delivery strategies for proteins and peptides. The design of these drug delivery systems in overcoming physical and chemical barriers along the gastrointestinal tract while improving oral bioavailability will be highlighted and discussed.

Novel Signal Peptides and Episomal Plasmid System for Enhanced Protein Secretion in Engineered Bacteroides Species.

The genus Bacteroides, a predominant group in the human gut microbiome, presents significant potential for microbiome engineering and the development of live biotherapeutics aimed at treating gut diseases. Despite its promising capabilities, tools for effectively engineering Bacteroides species have been limited. In our study, we have made a breakthrough by identifying novel signal peptides in Bacteroides thetaiotaomicron and Akkermansia muciniphila. These peptides facilitate efficient protein transport across cellular membranes in Bacteroides, a critical step for therapeutic applications. Additionally, we have developed an advanced episomal plasmid system. This system demonstrates superior protein secretion capabilities compared to traditional chromosomal integration plasmids, making it a vital tool for enhancing the delivery of therapeutic proteins in Bacteroides species. Initially, the stability of this episomal plasmid posed a challenge; however, we have overcome this by incorporating an essential gene-based selection system. This novel strategy not only ensures plasmid stability but also aligns with the growing need for antibiotic-free selection methods in clinical settings. Our work, therefore, not only provides a more robust secretion system for Bacteroides but also sets a new standard for the development of live biotherapeutics.

Chemical Modification of Cytochrome C for Acid-Responsive Intracellular Apoptotic Protein Delivery for Cancer Eradication.

Delivering bioactive proteins into cells without carriers presents significant challenges in biomedical applications due to limited cell membrane permeability and the need for targeted delivery. Here, we introduce a novel carrier-free method that addresses these challenges by chemically modifying proteins with an acid-responsive cell-penetrating peptide (CPP) for selective intracellular delivery within tumours. Cytochrome C, a protein known for inducing apoptosis, served as a model for intracellular delivery of therapeutic proteins for cancer treatment. The CPP was protected with 2,3-dimethyl maleic anhydride (DMA) and chemically conjugated onto the protein surface, creating an acid-responsive protein delivery system. In the acidic tumour microenvironment, DMA deprotects and exposes the positively charged CPP, enabling membrane penetration. Both in vitro and in vivo assays validated the pH-dependent shielding mechanism, demonstrating the modified cytochrome C could induce apoptosis in cancer cells in a pH-selective manner. These findings provide a promising new approach for carrier-free and tumour-targeted intracellular delivery of therapeutic proteins for a wide range of potential applications.

Ribonuclease A-polymer conjugates via in situ growth for cancer treatment.

Efficient delivery of therapeutic proteins is a critical aspect for protein-based cancer treatment. Herein, an in situ growth approach was employed to prepare ribonuclease A (RNase A)-polymer conjugates by incorporating a cationic polymer, poly(N,N'-dimethylamino-2-ethyl methacrylate) (P(DMAEMA)), and a hydrophobic polymer, poly(N-isopropylacrylamide) (P(NIPAM)), through atom transfer radical polymerization (ATRP). The synthesized RNase A-polymer conjugates (namely R-P(D-b-N)) could preserve the integrity of RNase A and exhibit a unique combination of cationic and hydrophobic properties, leading to enhanced intracellular delivery efficiency. The successful delivery of RNase A by R-P(D-b-N) conjugates effectively triggered the cell apoptosis through the mitochondria-dependent signaling pathway to achieve the anti-proliferative response. Additionally, the conjugates could inhibit cell migration and thus possess the potential for the suppression of tumor metastasis. Overall, our findings highlight that the introduction of cationic and hydrophobic moieties via ATRP provides a versatile platform for the intracellular delivery of therapeutic proteins, offering a new avenue for treating diverse diseases.

Photothermally Modulated Intracellular Delivery of Therapeutic Proteins Using Gold-Collagen Hybrid Nanoparticles

Photothermally Modulated Intracellular Delivery of Therapeutic Proteins Using Gold-Collagen Hybrid Nanoparticles

Eyedrop delivery of therapeutic proteins with zwitterionic polymers to treat dry age-related macular degeneration

In clinics, therapeutic proteins are commonly used to treat retinal diseases through intraocular injection, the treatment which suffers from rather low patient compliance. Topical administration (e.g. eye-drops) of large molecule drugs remains a major challenge due to the presence of various barriers in the eye. In this study, zwitterion-grafted chitosan (CS-ZW) was developed and then self-assembled with protein therapeutics including adalimumab (ADA) or catalase (CAT) for the treatment of dry age-related macular degeneration (dAMD) via topical eyedrops. Since CS-ZW can cross the mucus layer and open the tight junctions between epithelial cells, their delivered therapeutic proteins can be shuttled across the ocular barriers to reach the diseased site in the fundus. CS-ZW/ADA eyedrops delivering ADA to bind TNF-α in the fundus achieved a similar therapeutic effect to intravitreal ADA injection in a mouse dAMD model. In addition, the therapeutic effect was further improved by combining eyedrop formulations of CS-ZW/ADA and CS-ZW/CAT, the latter of which can clear reactive oxygen species (ROS) in the lesion to further assist dAMD treatment. Our work provides a simple and effective delivery vehicle that can non-invasively treat fundus diseases such as dAMD, showing potential advantages in reducing side effects associated with intraocular injection and improving patient compliance.

Enhanced neuroprotective activity of ophthalmic delivered nerve growth factor conjugated with cell penetrating peptide against optic nerve injury

Nerve growth factor is a well characterized neurotrophic factor that play a critical role in the survival, growth, and differentiation of neurons both in central and peripheral nervous system. However, it is difficult for the conventional exogenous nerve growth factor administration delivery to the central nervous system due to the biological barrier in human bodies. Here, we validated a series of cell penetrating peptides and found that L-PenetraMax significantly enhanced the efficiency of recombinant human nerve growth factor entry into the rat retina. In the optic nerve crush mice model, eye drop administration of recombinant human nerve growth factor alone promoted retinal ganglion cell survival and axon regeneration at high dose, while the combination of recombinant human nerve growth factor with L-PenetraMax significantly enhanced the neuroprotective efficacy at lower dose, thus potentially enhancing the availability of recombinant human nerve growth factor eye drops in patients with optic neuropathy. This study provides the evidence that the noncovalent coadministration of recombinant human nerve growth factor with L-PenetraMax could be a potent strategy for the non-invasive and sustained ocular delivery of therapeutic proteins for improving the optic nerve injury.

Recent Developments in Gene Therapy for Neovascular Age-Related Macular Degeneration: A Review.

Age-related macular degeneration (AMD) is a complex and multifactorial disease and a leading cause of irreversible blindness in the elderly population. The anti-vascular endothelial growth factor (anti-VEGF) therapy has revolutionized the management and prognosis of neovascular AMD (nAMD) and is currently the standard of care for this disease. However, patients are required to receive repeated injections, imposing substantial social and economic burdens. The implementation of gene therapy methods to achieve sustained delivery of various therapeutic proteins holds the promise of a single treatment that could ameliorate the treatment challenges associated with chronic intravitreal therapy, and potentially improve visual outcomes. Several early-phase trials are currently underway, evaluating the safety and efficacy of gene therapy for nAMD; however, areas of controversy persist, including the therapeutic target, route of administration, and potential safety issues. In this review, we assess the evolution of gene therapy for nAMD and summarize several preclinical and early-stage clinical trials, exploring challenges and future directions.

Role of Polymer Concentration on the Release Rates of Proteins from Single- and Double-Network Hydrogels

Controlled delivery of proteins has immense potential for the treatment of various human diseases, but effective strategies for their delivery are required before this potential can be fully realized. Recent research has identified hydrogels as a promising option for the controlled delivery of therapeutic proteins, owing to their ability to respond to diverse chemical and biological stimuli, as well as their customizable properties that allow for desired delivery rates. This study utilized alginate and chitosan as model polymers to investigate the effects of hydrogel properties on protein release rates. The results demonstrated that polymer properties, concentration, and crosslinking density, as well as their responses to pH, can be tailored to regulate protein release rates. The study also revealed that hydrogels may be combined to create double-network hydrogels to provide an additional metric to control protein release rates. Furthermore, the hydrogel scaffolds were also found to preserve the long-term function and structure of encapsulated proteins before their release from the hydrogels. In conclusion, this research demonstrates the significance of integrating porosity and response to stimuli as orthogonal control parameters when designing hydrogel-based scaffolds for therapeutic protein release.

Advancing Oro-Dental and Cranio-Maxillo-Facial Interventions for QoL: Injectable Hydrogel and BioCeramic NanoDelivery Platforms

In the ever-evolving landscape of healthcare, where science, technology, and clinical practice converge, the realm of cranio-maxillo-facial and oro-dental interventions stands at the forefront of transformative possibilities. This communication embarks on an illuminating journey into the heart of this dynamic field, where innovation knows no bounds. Collaborative student engagement has been instrumental in not only exploring but also bridging the chasm between diverse scientific disciplines and the practical intricacies of clinical application. Recent strides in drug delivery mechanisms, characterized by the ingenious fusion of cells and nano-carriers, have laid the foundation for a revolution in regenerative medicine. These breakthroughs promise a future where treatments are not only precise but also meticulously controlled, enhancing patient quality of life through the amelioration of disease conditions and restoration of bodily functions. Injectable hydrogels, another trailblazing innovation, have emerged as a beacon of hope. They offer versatile solutions that promise to redefine surgical and clinical practices by affording healthcare professionals spatio-temporal control, tunability, and stimuli-responsiveness. These hydrogels unlock the potential for the targeted delivery of therapeutic agents, including cells, genes, proteins, and drugs, revolutionizing tissue engineering, regenerative medicine, and aesthetic surgical interventions. BioCeramics, a burgeoning field, is redefining conventional wisdom in bone grafting and implant technology. These biomaterials are poised to usher in a new era of innovative approaches that enhance the mechanical and biological compatibility of implants, facilitating improved clinical outcomes and patient quality of life. Moreover, this communication serves as a herald of exciting news: the inauguration of a Doctoral (PhD) Program in Dental Sciences at the prestigious Universidad de los Andes in Santiago de Chile. This program beckons forward-thinkers, visionaries, and emerging scholars to partake in pioneering research at the Laboratorio de Biomateriales, Farmace uticos y Bioingenierí a de Tejidos Cra neo Ma xilo-Facial. As we embrace the future, limitless possibilities abound in this transformative health field, where advancing science and technology merge seamlessly to enhance the clinical and surgical armamentarium available for contemporary practices and users. This journey represents not only a dedication to scientific exploration yet an unwavering commitment to improving patient quality of life (QoL) and well-being thru the amalgamation of cutting-edge research, innovation, and compassionate care.

B Cell Therapeutics

Plasma cells are specialized immune cells that can survive in the bone marrow for decades and continually secrete large amounts of antibodies, thus providing long-term, often lifelong protection from repeated exposures to a pathogen. The long-term survival and the capability to secrete high amounts of protein make plasma cells a potential candidate for stable delivery of therapeutic proteins and antibodies. However, engineering plasma cells or B cells has been challenging due to low lentiviral transduction efficiency, incompatibility with plasmid transfection and loss of episomal DNA expression over time. We have previously shown that human plasma cells can be engineered to secrete antibodies and proteins in in vitro culture systems via gene editing. In the current study, we have generated engineered murine plasma cells that can secrete the clotting factor IX (FIX). We hypothesize that the FIX-secreting plasma cells will engraft long-term in FIX-deficient immunocompetent mice and secrete therapeutic levels of FIX leading to protection from Hemophilia B. The standard of care for Hemophilia B is repeated intravenous infusion of recombinant FIX or AAV-based gene therapy. However, 30-60% humans have pre-existing immunity to AAV-based gene therapy and gene therapy can induce an immune response in people lacking pre-existing antibodies. Moreover, gene therapy is unsuitable for pediatric patients due to their rapid growth and proportional loss of AAV. Plasma cell-based therapy is lowly immunogenic and can be used for redosing in pediatric patients that require more product with age. Here, we will describe proof-of-concept studies showing that engineered murine plasma cells can engraft into immunocompetent mice without the need for conditioning and produce stable levels of protein for >10 weeks. We developed a three-step in vitro co-culture system that can be used to engineer and differentiate naïve B cells into plasma cells. These in vitro generated murine plasma cells had a comparable gene expression profile and phenotype to natural plasma cells and can engraft stably, in the absence of conditioning, into immunocompetent mice whereupon they secreted antibodies (Figure 1). Moreover, we show that plasma cells can be re-dosed to increase antibody levels without triggering a secondary immune response (Figure 1). For delivery of therapeutic proteins, we have developed adeno-associated virus-based homology directed repair reagents to edit murine B cells at several loci, including beta-2 microglobulin, Rosa-26, and the antibody heavy chain. By co-delivery of cis-linked fluorescent markers, we were able to sort and expand only edited cells, enabling enrichment of cells expressing a therapeutic protein. In proof-of-concept studies, we have used this approach to generate a human FIX-secreting plasma cell product with close to 90% edited cells at the end of the B cell culture. The enriched plasma cells generated 0.28 pg FIX/cell/day compared to 0.19 pg FIX/cell/day generated by the bulk edited cells (figure 2). We estimate that engraftment of 1 million edited plasma cells is sufficient to generate therapeutic levels of FIX upon (figure 2). We have developed reagents for a novel cell-based therapeutic tool with protein secretion capabilities and far-reaching applications. Out studies will provide insights into the feasibility and efficacy of a B cell based therapeutic product.