Zielinska N, Howard EL, Stevens BAY
… +18 more, Goens MM, Campbell ESB, Hughes ME, Pei Y, Xu L, Achuthan A, Vadivel A, Apostol A, He X, Zheng D, Gordon DB, Srinivas RR, Nielsen AAK, Caswell JL, Arroyo LG, Yuen DA, Thebaud B, Wootton SK
Recombinant adeno-associated virus (rAAV) platforms have achieved significant success in clinical gene therapy; however, many still rely on ubiquitous promoters. This robust and widespread transgene expression can cause...Recombinant adeno-associated virus (rAAV) platforms have achieved significant success in clinical gene therapy; however, many still rely on ubiquitous promoters. This robust and widespread transgene expression can cause off-target effects, immune activation, and systemic toxicity, limiting their suitability for diseases requiring tissue-specific expression, such as surfactant protein B (SP-B) deficiency. Here, we aimed to improve the precision of AAV-lung gene therapy by evaluating computationally predicted lung-specific promoters with AAV6.2FF, a capsid with strong lung tropism. Promoter strength and specificity were assessed following administration of AAV6.2FF encoding the human placental alkaline phosphatase (AP) reporter gene in mice. Cross-species activity was evaluated in precision-cut lung slices (PCLS) from ferrets and pigs. We identified promoter 5979 as lung-specific in mice, outperforming the ubiquitous CASI promoter (comprised of the cytomegalovirus enhancer, chicken β-actin promoter, and ubiquitin C regulatory elements) in transgene expression and specificity, independent of AAV capsid or route of administration. In a conditional SP-B knockout model, AAV6.2FF-5979-hSPB extended survival in SP-B-deficient mice and outperformed its CASI-driven counterpart. Promoter 5979 exhibited the highest activity among the four synthetic promoters in ferret PCLS, but demonstrated limited activity in pig PCLS, underscoring species-specific differences. This study demonstrates the therapeutic value of tissue-specific promoters in targeted gene therapy while emphasizing the importance of refining algorithmic prediction platforms to ensure reliable cross-species and clinical performance.
Adeno-associated virus (AAV) is widely accepted as a delivery vector for in vivo gene therapy due to its relatively low immunogenicity, minimal toxicity, sustained efficacy, and broad tropism. However, its unpredictable...Adeno-associated virus (AAV) is widely accepted as a delivery vector for in vivo gene therapy due to its relatively low immunogenicity, minimal toxicity, sustained efficacy, and broad tropism. However, its unpredictable cross-species applicability remains a troublesome hurdle for broader clinical applications. Thus, designing novel AAV capsids with enhanced cross-species applicability is urgently needed. In this review, we present AAV bioengineering methods, including rational design, directed evolution, and artificial intelligence-based design, with the goal of creating novel AAV variants that are translatable to humans. Using representative examples, we also evaluate how each method addresses key species-dependent barriers-receptor usage, intracellular trafficking, immune recognition, and toxicity-that critically determine cross-species translatability.
In pursuit of a gene transfer agent with efficient pulmonary transduction, the UK Respiratory Gene Therapy Consortium has developed a lentiviral vector pseudotyped with the envelope proteins, F and HN from Sendai virus (...In pursuit of a gene transfer agent with efficient pulmonary transduction, the UK Respiratory Gene Therapy Consortium has developed a lentiviral vector pseudotyped with the envelope proteins, F and HN from Sendai virus (rSIV.F/HN). In contrast to other viral vectors, pulmonary rSIV.F/HN delivery achieves sustained gene expression ( ~ 2 years in mice) in the lungs and systemic circulation following a single dose. Here, we investigate the application of the rSIV.F/HN vector-platform for wider indications, including systemic disorders that require serum expression of therapeutic proteins. To assess the potential for rSIV.F/HN to produce systemic proteins, intravenous vector delivery was characterised and compared against intrapulmonary administration, achieved via 'nasal sniffing'. Both delivery routes achieved sustained (at least 1 year) systemic expression of the secreted reporter protein Gaussia luciferase. Systemic rSIV.F/HN delivery resulted in widespread protein expression across multiple organs, accompanied by the generation of significant anti-vector neutralising antibodies limiting vector readministration. Conversely, localised airway transduction was observed following pulmonary administration, which we have previously shown is not an impediment to efficient vector readministration. These data support intrapulmonary rSIV.F/HN delivery for systemic protein production, with sustained high-level transgene expression and feasible readministration.
Genome editing has progressed from a laboratory capability for targeted DNA manipulation to a clinically relevant strategy for correcting, silencing, or regulating genes implicated in human disease. In this Review, we sy...Genome editing has progressed from a laboratory capability for targeted DNA manipulation to a clinically relevant strategy for correcting, silencing, or regulating genes implicated in human disease. In this Review, we synthesize the mechanisms, capabilities, and constraints of the principal programmable platforms-zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas systems-and highlight how base editors, prime editors, and epigenetic editors expand the range of achievable outcomes beyond double-strand break-dependent repair to precise nucleotide substitutions, small insertions/deletions, and transcriptional modulation. We compare genome-editing cargo formats, including plasmid DNA, viral-vector DNA, mRNA, guide RNA, and ribonucleoprotein complexes, together with the delivery modalities used to transport them, including AAV, adenoviral and herpesviral vectors, lipid nanoparticles (LNPs), electroporation, and virus-like particles. We then consolidate key biomedical applications enabled by these technologies, spanning endogenous gene tagging, high-throughput functional variant screening, molecular recording, and the generation of genetically faithful disease models. Across oncology, respiratory, hematologic, cardiovascular, metabolic, neurodegenerative, viral, ocular, and immune disorders, genome editing is advancing both ex vivo and in vivo interventions, including engineered cellular immunotherapies, hematopoietic stem and progenitor cell editing for hemoglobinopathies, and emerging liver-directed programs for lipid and coagulation targets. Finally, we discuss priorities for broad clinical implementation: improving editing fidelity and PAM flexibility, increasing performance in non-dividing cells, enabling tissue-selective delivery to difficult organs (for example, lung and central nervous system), and addressing manufacturing scalability, long-term monitoring, and equitable global access.
Recombinant Adeno-Associated Virus (rAAV) is the leading viral vector platform for gene therapy. A persistent challenge in rAAV manufacturing and quality control is accurate assessment of the DNA content of purified vira...Recombinant Adeno-Associated Virus (rAAV) is the leading viral vector platform for gene therapy. A persistent challenge in rAAV manufacturing and quality control is accurate assessment of the DNA content of purified viral capsids. Empty particles, as well as 'partial' and over-filled species commonly contaminate even highly purified rAAV preparations. These impurities contribute to high production costs and reduced therapeutic efficacy, resulting in higher required doses and increased risks to patient health. Here we present high-resolution electrospray differential mobility analysis (ES-DMA) as a novel benchtop analytical modality for rAAV vector characterization. Sampling microliter volumes of analyte, this method achieves angstrom-scale particle sizing which we demonstrate is sufficient to resolve DNA encapsidation by rAAVs via differential electrical mobility. We benchmark this approach against charge detection mass spectrometry (CD-MS), an emerging gold standard for rAAV analytics, and identify a robust near-linear correlation between electrical mobility and mass of aerosolized rAAV particles. This relationship enables rapid identification and relative quantification of empty, full, and partial / over-filled rAAV capsids by ES-DMA.
Hereditary protein C (PC) deficiency, which is caused by PROC gene mutations, increases the risk of venous thromboembolism and offers limited treatment options. In this study, we developed adeno-associated virus serotype...Hereditary protein C (PC) deficiency, which is caused by PROC gene mutations, increases the risk of venous thromboembolism and offers limited treatment options. In this study, we developed adeno-associated virus serotype 8 (AAV8) vectors carrying either murine PC (AAV8-mPROC) or human PC (AAV8-hPROC) transgenes. These vectors were delivered through tail vein injection into PROC knockout mice. The highest dose of AAV8-mPROC (6.00E + 12 vg/kg) resulted in PC activity and antigen levels reaching 200.7% and 190.1%, respectively, which were maintained at 171.8% and 165.2%, respectively, by week 48. Similarly, the highest dose of AAV8-hPROC (8.00E + 12 vg/kg) resulted in 295.4% PC activity and 3.72 μg/ml human protein C antigen, which were maintained at 195.1% and 1.13 μg/ml, respectively, by week 48. In the vein thrombosis model, AAV8-mPROC and AAV8-hPROC significantly reduced the thrombus weight from 12.11 ± 3.39 mg to 7.19 ± 2.28 mg and 6.81 ± 2.28 mg, respectively. In the pulmonary embolism model, the proportion of embolized vessels decreased from 88.53% to approximately 60.62% in the AAV8-mPROC group and 62.33% in the AAV8-hPROC group. Our study has established a preclinical foundation for the safe and effective application of AAV vector-based gene therapy in treating inherited PC deficiency.
Use of adeno-associated virus (AAV) vectors has revolutionized in vivo gene therapy, but the presence of pre-existing neutralizing antibodies remains a major barrier that can hinder clinical application. While large-anim...Use of adeno-associated virus (AAV) vectors has revolutionized in vivo gene therapy, but the presence of pre-existing neutralizing antibodies remains a major barrier that can hinder clinical application. While large-animal models such as non-human primates have been used to study anti-AAV immunity, their high cost and limited accessibility present challenges for studying the impact of AAV immunity on AAV-based therapies. Here, we evaluate pigs as an immunologically relevant large-animal model for investigating humoral barriers to AAV-based gene therapy. Using ELISA-based profiling across 11 AAV serotypes, we detected immunoglobulin G (IgG) antibodies against AAV capsids in pigs as early as two weeks of age, with titers increasing with age and displaying serotype-specific dynamics. Animals maintained in standard housing displayed greater inter-individual variations and broader serotype-specific reactivities. Functional assays demonstrated that these antibodies neutralized AAV particles in a dose- and serotype-dependent manner, with IgG depletion restoring transduction in vitro. Sequence analysis indicated that capsid identity can partially predict cross-reactive binding, but only under controlled conditions. These findings establish pigs as a tunable model capable of recapitulating age- and environment-dependent features of anti-AAV immunity, providing a platform for studying humoral barriers and evaluating immune evasion strategies in AAV-based gene therapy.
Chimeric antigen receptor (CAR) T cell therapies have shown remarkable success in the treatment of hematologic cancers; however, their use is often accompanied by inflammatory toxicities, including cytokine release syndr...Chimeric antigen receptor (CAR) T cell therapies have shown remarkable success in the treatment of hematologic cancers; however, their use is often accompanied by inflammatory toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). These toxicities, ranging from mild to life-threatening, are partly driven by bystander myeloid cell activation (BMCA) and the subsequent release of pro-inflammatory cytokines such as IL-6 and IL-1β. Although previous studies have described the individual contributions of GM-CSF, IFN-γ, and TNFα secreted by CAR-T cells, a comprehensive characterization of CAR-T-derived inflammatory factors has been lacking. In this study, we characterized the soluble factors secreted by activated CAR-T cells derived from human peripheral blood and assessed their role in BMCA. Comparative cytokine analyses across human T cell subsets, including CAR-T cells, identified multiple candidates involved in BMCA. Antibody-mediated neutralization confirmed that four factors, GM-CSF, IFN-γ, TNFα, and GPIbα, play dominant roles in driving BMCA. Furthermore, siRNA-mediated knockdown of these factors in CAR-T cells significantly reduced BMCA without impairing their anti-tumor activity. These findings are consistent with prior reports on the inflammatory roles of GM-CSF, IFN-γ, and TNFα during CAR-T cell therapy and, importantly, identify GPIbα as a previously unrecognized contributor to CAR-T-associated inflammatory toxicities. Targeting these factors through antibody blockade or genetic modification may represent a promising strategy to mitigate inflammatory toxicities and improve the safety of CAR-T cell therapies.
The inhibition of estrogen receptor (ER)-mediated genomic signaling in ER-positive cancer cells has long been a primary focus of therapeutic strategies. Here, we introduce a switchable competitive inhibition system for E...The inhibition of estrogen receptor (ER)-mediated genomic signaling in ER-positive cancer cells has long been a primary focus of therapeutic strategies. Here, we introduce a switchable competitive inhibition system for ERα-mediated transcriptional regulation, termed DOCTER (Drug-induced On-Off Competitor for Transcription mediated by ERα). DOCTER integrates the Tet-On induced Cre-loxP recombination system to enable precise, reversible on/off switching. We demonstrate that DOCTER effectively inhibits ERα-mediated transcriptional regulation in breast cancer cells, modulating both exogenous and endogenous gene expression, and remains effective in cells harboring ERα ligand-binding domain (LBD) mutations. Upon drug induction, DOCTER exhibits controllable and reversible inhibition. To visualize these dynamic switching events in real time, we developed a multi-color fluorescent reporting system that enables monitoring of complete DOCTER switch-off within 24 hours. Our study provides a novel approach for time-specific transcriptional regulation and offers broad potential for applications in genetic research and therapeutic development.
Antibody-oligonucleotide conjugates (AOCs) effectively integrate the delivery capability of antibodies with the specific gene regulatory function of oligonucleotides, offering a novel strategy for extrahepatic delivery....Antibody-oligonucleotide conjugates (AOCs) effectively integrate the delivery capability of antibodies with the specific gene regulatory function of oligonucleotides, offering a novel strategy for extrahepatic delivery. Unlike antibody-drug conjugates (ADCs), AOCs employ nucleic acid payloads, enabling specific gene modulation with reduced off-target effects, thus providing new avenues for treating genetic disorders. While AOC candidates for conditions such as Duchenne muscular dystrophy (DMD) have progressed to Phase III trials, their development remains constrained by limited targets like TfR1, and their activity is highly dependent on antibody selection, linker design, and modification strategies. This study delves into the fundamental principles of target selection and antibody engineering, emphasizing that the endocytic efficiency of the target receptor is a critical factor for the successful delivery of AOC therapeutics. Taking TfR1 as an example, it analyzes the advantages and disadvantages of different antibody formats, examines the structure-activity relationships between linker chemistry and pharmacokinetics/pharmacodynamics, and further explores nucleic acid modification strategies aimed at enhancing delivery efficiency. Finally, the study outlines future directions for AOC development, including advances in bispecific antibodies, peptide conjugation, gene editing, and artificial intelligence-driven approaches, aiming to provide forward-looking perspectives and a theoretical foundation for the design of AOC therapeutics.
Hemophilia A (HA), an X-linked bleeding disorder caused by factor VIII (FVIII) deficiency, is primarily managed with exogenous therapeutic agents; however, this treatment approach remains burdensome and fails to provide...Hemophilia A (HA), an X-linked bleeding disorder caused by factor VIII (FVIII) deficiency, is primarily managed with exogenous therapeutic agents; however, this treatment approach remains burdensome and fails to provide durable hemostatic control. Adeno-associated viral (AAV) vectors enabling endogenous FVIII expression have emerged as promising alternatives to address these limitations, but existing vectors show limited transduction efficiency and declining activity over time. Here, we report the development and preclinical evaluation of two bioengineered AAV8 vectors, HMR-001 and its codon-optimized variant HMR-001z, designed to enhance genome integrity, hepatocellular delivery, and translational efficiency. In hemophilia A mice, intravenous administration of HMR-001 induced dose-dependent and sustained FVIII expression, achieving substantial hemostatic improvement at the highest investigated dose (2 × 10¹³ vg/kg), with blood loss reduced to levels comparable to Xyntha prophylaxis. High-order triple-linkage ddPCR quantification revealed dose-dependent increases in full-length vector genome abundance, reaching 3.74, 21.55, and 48.52 copies per diploid genome at doses of 2 × 10¹², 8 × 10¹², and 2 × 10¹³ vg/kg, respectively. Building on these markedly improved genome delivery and preservation profiles, HMR-001z achieved complete hemostatic correction at a dose of 1 × 10¹³ vg/kg, exhibiting approximately 30-fold higher FVIII expression than HMR-001 and reaching ~400 IU/dL. Collectively, these findings demonstrate that genome-level optimization combined with codon-usage refinement synergistically enhances AAV8-mediated FVIII expression, establishing HMR-001z as a durable and translationally advanced gene therapy candidate for hemophilia A.
Charcot-Marie-Tooth disease type 4C is a demyelinating neuropathy caused by loss of function mutations in the SH3TC2 gene, that is highly expressed in myelinating Schwann cells. We generated and tested a clinical stage v...Charcot-Marie-Tooth disease type 4C is a demyelinating neuropathy caused by loss of function mutations in the SH3TC2 gene, that is highly expressed in myelinating Schwann cells. We generated and tested a clinical stage vector with a minimal human MPZ promoter driving expression of SH3TC2. Groups of 1-month old Sh3tc2 mice were treated with 3 different doses of AAV9-hMPZmini.SH3TC2.SV40pA or the formulation buffer by lumbar intrathecal injection. Outcomes were compared 8 weeks post injection by behavioral, electrophysiological, proteomics, morphological analysis and evaluation of tissue integrity and inflammatory responses. Vector biodistribution to the peripheral nerves and high rates of cell-specific therapeutic gene expression in Schwann cells resulted in significant therapeutic benefits in the CMT4C model. Treated mice showed improved motor performance in grip strength and motor nerve conduction velocities. Morphological analysis revealed significant improvement in g-ratios, myelin thickness and ratios of demyelinated fibers in lumbar roots and femoral nerves of treated mice. Proteomic profiles showed correction of muscle denervation associated pathobiochemical processes in treated mice. Not observed tissue toxicity or immune reactions in neural tissues or peripheral organs. This study provides proof of principle for dose-dependent effectiveness and safety of intrathecal AAV9-mediated gene replacement paving the way for clinical translation.
The benefit of early diagnosis and treatment has been demonstrated in animal models of several lysosomal storage diseases. In a clinical setting, however, diagnoses are often not made until after patients become symptoma...The benefit of early diagnosis and treatment has been demonstrated in animal models of several lysosomal storage diseases. In a clinical setting, however, diagnoses are often not made until after patients become symptomatic. The lysosomal storage disease alpha-mannosidosis is caused by a genetic deficiency of lysosomal alpha-mannosidase, leading to the widespread presence of storage lesions throughout the brain and other tissues. In a feline model of alpha-mannosidosis, we previously demonstrated complete correction of the brain following delivery of AAVhu.32-fMANB via the carotid artery in the early symptomatic stage. Here, we investigate the efficacy of AAV gene therapy on globally distributed storage lesions in animals with advanced disease. Some improvements in clinical parameters were observed, however these improvements were less than in animals with less advanced disease. Although the treated animals were improved compared to untreated animals, increasing the vector dose did not further improve clinical outcomes. These results further demonstrate the importance of early detection and treatment of a lysosomal storage disease to successful outcomes. Despite this, partial correction extended the lifespan of diseased cats and may be medically beneficial to patients by slowing or stabilizing the progressive degenerative course of disease.
Adeno-associated virus serotype 2 (AAV2) remains one of the most common vectors for CNS gene delivery. Yet, its long-term intraparenchymal trafficking and the relationship between capsid persistence and transgene express...Adeno-associated virus serotype 2 (AAV2) remains one of the most common vectors for CNS gene delivery. Yet, its long-term intraparenchymal trafficking and the relationship between capsid persistence and transgene expression remain poorly understood. In this study, we tracked the spatial and temporal patterns of AAV2 following direct striatal infusion in rats, analyzing tissue at 3 days, 3 weeks, 12 weeks, 30 weeks, and 67 weeks after injection. Using immunofluorescence for the human aromatic L-amino acid decarboxylase (AADC) transgene and an epitope detecting intact AAV2 capsids (A20), we mapped capsid localization, clearance, and axonal transport over more than a year. Following direct striatal infusion, AAV2 capsids rapidly localized to striatal neurons, with early accumulation in the substantia nigra pars reticulata (SNpr) detectable by 3 days post-infusion, but without evidence of nigral neuron transduction. Transgene expression increased over time and peaked locally at 12 weeks, with a delayed yet robust AADC signal in striatonigral terminals by 30 weeks. By 67 weeks, capsid signal was minimal while AADC expression remained stable. These findings clarify the long-term dynamics of AAV2 distribution, capsid persistence, and axonal transport in the adult brain, informing our understanding of vector behavior and durability following intraparenchymal AAV2 gene delivery.
The IVS4+919G>A mutation in the GLA gene, prevalent in East Asian populations, causes cardiac-type Fabry disease by creating an abnormal splice site. This results in the insertion of a 57-nucleotide segment between exon...The IVS4+919G>A mutation in the GLA gene, prevalent in East Asian populations, causes cardiac-type Fabry disease by creating an abnormal splice site. This results in the insertion of a 57-nucleotide segment between exon 4 and exon 5, introducing a premature stop codon and leading to a truncated, non-functional α-Gal A protein. We evaluated whether adenine base editing (ABEmax) can modulate this allele-induced cryptic splice event in patient-derived fibroblasts in vitro as a proof-of-concept. Two ABEmax/sgRNA constructs targeting intron 4 (ABEmax-sgRNA1 and ABEmax-sgRNA2) were tested; both induced on-target +919 A → G conversion with frequent bystander edits at +918/+920. Edited bulk populations and single-cell-derived clones showed restoration of correctly spliced GLA mRNA with reduced aberrant transcripts, increased GLA protein, higher α-Gal A activity (approaching wild-type levels in some clones), and reduced intracellular Gb3 signal. A focused next-generation sequencing panel identified a low-frequency intronic change at one predicted off-target locus without predicted coding consequences. These findings demonstrate in vitro splice rescue of a deep intronic, cardiac-type Fabry disease variant by adenine base editing and suggest that bystander edits in non-coding sequence can further enhance correction by suppressing cryptic splicing, with concordant improvements in α-Gal A activity and Gb3 signals.
The innovative clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease 9 (Cas9) gene editing technique may represent a suitable therapeutic opportunity for the treatment of inherited diseas...The innovative clustered regularly interspaced short palindromic repeats (CRISPR) associated nuclease 9 (Cas9) gene editing technique may represent a suitable therapeutic opportunity for the treatment of inherited diseases such as Wilson disease (WD). This monogenetic liver disease is based on a mutation of the ATP7B gene and leads to a functional deterioration in copper (Cu) excretion. Excess Cu accumulations in organs such as the liver and brain lead to severe cytotoxicity, followed by acute or chronic liver failure and/or neurological symptoms, and even death, which makes cellular Cu excretion indispensable for any potential WD therapy, e.g., gene therapy. A life-long treatment with zinc or chelators such as D-penicillamine may improve the course of the disease, but serious side effects have been observed in a significant portion of patients. In this study, isolated urinary epithelial cells from a WD patient carrying the ATP7B H1069Q mutation were reprogrammed into induced pluripotent stem cells (iPSCs). Using the CRISPR/Cas9 technology, ATP7B H1069Q was corrected by the additional use of single-stranded oligo DNA nucleotides (ssODNs). After differentiation into hepatocyte-like cells (HLCs), a high resistance to Cu was observed, plus a recovery of ATP7B trafficking. This is the first study to confirm that CRISPR/Cas9-mediated correction of the ATP7B point mutation H1069Q is possible and could open new possibilities for future applications.
Antimicrobial resistance (AMR) is one of the most serious and pressing health challenges facing modern medicine. Despite advances in antimicrobial stewardship, diagnostics and infection prevention, the rapid emergence an...Antimicrobial resistance (AMR) is one of the most serious and pressing health challenges facing modern medicine. Despite advances in antimicrobial stewardship, diagnostics and infection prevention, the rapid emergence and spread of resistant pathogens continues to limit treatment options and increase morbidity, mortality and healthcare costs. The discovery of new innovative antimicrobial therapies remains of paramount importance. In this context, gene therapy is gaining attention as a complementary strategy that can directly target the molecular genetic determinants of antimicrobial resistance. Recent advances in RNA-based technologies and CRISPR-Cas systems have enabled increasingly precise manipulation of microbial genomes, opening up the possibility of restoring antimicrobial susceptibility, reducing virulence and limiting the spread of resistance genes.
Tenuta M, Bravo M, Olson A
… +13 more, Saney CL, Weinfurter J, Ben-Akiva E, Bhange D, Cottrell CA, Vosler L, Weisgrau K, Burton DR, Irvine DJ, Schief WR, Rakasz E, Joyner CJ, Voss JE
HIV broadly neutralizing antibodies (bnAbs) are promising reagents for prevention and therapy of disease; however, their elicitation is constrained by genetic limitations of the human B cell antigen-receptor (BCR) repert...HIV broadly neutralizing antibodies (bnAbs) are promising reagents for prevention and therapy of disease; however, their elicitation is constrained by genetic limitations of the human B cell antigen-receptor (BCR) repertoire. Precision genome-editing offers a potential solution by enabling bnAb genes to be programmed into the BCR repertoire as IgH-modified B cells. Such cells can be vaccinated to elicit durable bnAb memory responses in mice; however, extending this success to non-human primates (NHPs) would be a major advance towards clinical translation. Here, we show that ex vivo reprogrammed NHP B cells can survive autologous infusion and respond to immunization, differentiating into antibody-secreting cells (ASCs) that can produce up to 1 µg/ml of a bnAb in serum following vaccination prime. Although durable transgenic memory responses were not generated, vaccination of engineered cells in secondary lymphoid organoid (SLO) cultures recapitulated transient ASC responses in vitro. These findings suggest that NHP-derived SLOs could provide a platform to optimize engineering and vaccination conditions that drive germinal center maturation of IgH-reprogrammed B cells in a clinically relevant NHP model, supporting the development of engineered B-cell vaccines that generate durable bnAb responses as a potential functional cure for HIV.