Effective scar control requires selectively suppressing late-stage fibrosis without compromising early wound closure. We developed a localized, time-staged delivery system. A poly-(HA-GMA) hydrogel serves as a short-term...Effective scar control requires selectively suppressing late-stage fibrosis without compromising early wound closure. We developed a localized, time-staged delivery system. A poly-(HA-GMA) hydrogel serves as a short-term depot, loaded with AAV8-sTβRII and applied directly along the wound margin. Materials characterization showed a water-rich porous network that rapidly imbibes and releases vector primarily by diffusion. In vivo, the hydrogel naturally degrades in ~3 days, enabling local enrichment in early healing without excessive retention. In a mouse full-thickness skin-wound model, this approach achieved efficient transduction of the cutaneous and fascial layers while markedly reducing hepatic exposure. From postoperative day 6 onward it accelerated closure and produced a thinner dermis, more orderly collagen organization, and a lower collagen area fraction. Mechanistically, Flag-sTβRII was detected within scar tissue. Phospho-Smad2/3 and α-SMA were reduced, whereas total Smad2/3 was largely unchanged, indicating that inhibition occurs at the activation step of the TGF-β/Smad pathway. Moreover, adding exogenous TGF-β1 reversed the macroscopic and histological benefits, strengthening the evidence for pathway specificity. Compared with direct intradermal injection, hydrogel delivery simultaneously increased local expression and limited systemic spillover. Using the AAV8 capsid provided the most favorable balance-high in skin, low in liver. Safety readouts-including body weight, serum transaminases, and histology of major organs-showed no abnormalities. To our knowledge, the "HA-GMA × AAV8-sTβRII" strategy precisely aligns pathway antagonism with the escalation phase of fibrosis, yielding improvements from molecular and cellular phenotypes to tissue remodeling and healing. It offers a generalizable, materials-biology integrated platform for anti-fibrotic gene therapy at the wound edge.
Gene-based editing can potentially correct the genetic defect in methylmalonic acidemia (MMA). SUNRISE, a first-in-human phase 1/2 open-label study, evaluated the safety/tolerability (primary endpoints) of liver-targeted...Gene-based editing can potentially correct the genetic defect in methylmalonic acidemia (MMA). SUNRISE, a first-in-human phase 1/2 open-label study, evaluated the safety/tolerability (primary endpoints) of liver-targeted hLB-001 in four pediatric participants (ages 20-114 months) with mitochondrial methylmalonyl-CoA mutase (MMUT)-deficient MMA. We designed a single-infusion adeno-associated viral capsid (hLB-001) to nondisruptively integrate functional MMUT at the 3' end of the albumin (ALB) locus to produce both albumin and MMUT. All four participants experienced at least one treatment-emergent adverse event. Three participants had treatment-emergent serious adverse events of cytokine release syndrome (one participant) and thrombotic microangiopathy (two participants); all resolved during the trial. Biologic activity, clinical efficacy, and 1-year survival were secondary endpoints. MMUT expression (measured by 2A-tagged ALB biomarker expression) increased in two participants over two years, confirming homology-based integration and positive selection of transgenic cells. However, serum methylmalonic acid (sMMA), serum FGF21, serum methylcitric acid (sMCA), and propionate oxidation remained abnormal in all four participants. All participants were alive at 1 year and at database lock. SUNRISE was terminated due to lack of efficacy. These results provide proof-of-concept for use of liver-targeted gene editing without nucleases for MMA and other genetic metabolic disorders. ClinicalTrials.gov identifier: NCT04581785Target journal: Gene Therapy (Springer Nature).
Messenger RNA therapeutics offer broad potential across various diseases, yet achieving sustained and efficient protein expression remains a central challenge. In this study, we report CJ-1, a novel mRNA construct engine...Messenger RNA therapeutics offer broad potential across various diseases, yet achieving sustained and efficient protein expression remains a central challenge. In this study, we report CJ-1, a novel mRNA construct engineered through systematic optimization of major regulatory elements, including the 5' and 3' untranslated regions and poly (A) tail. CJ-1 consistently outperformed first-generation mRNA constructs in protein expression across multiple cell types and in vivo mouse models. Moreover, CJ-1 elicited markedly lower cytokine responses, indicating reduced innate immune activation. To evaluate its therapeutic applicability, erythropoietin (EPO)-encoding CJ-1 mRNA was encapsulated in a Pfizer-BioNTech lipid nanoparticle formulation and administered intraperitoneally in mice. This resulted in elevated, sustained serum EPO levels and significant increases in reticulocyte counts and hematocrit. These findings support CJ-1 as a promising mRNA platform with enhanced expression and minimal immunogenicity, advancing the development of safer and more effective mRNA-based therapies.
As more advanced cell and gene therapies, including gene editing technologies, progress through drug development, there is increased emphasis on the importance of stakeholders, including people living with disease, careg...As more advanced cell and gene therapies, including gene editing technologies, progress through drug development, there is increased emphasis on the importance of stakeholders, including people living with disease, caregivers, and healthcare professionals, to communicate using clear, accurate, and consistent language. Lexicons explaining advanced gene therapies will support patients' and clinicians' understanding, enabling shared decision-making and informed consent for clinical trial participation and, in the future, healthcare choice. Early lexicon development is crucial for standardizing communication across clinical sites, geographies, clinicians, and patients. A lexicon for clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) gene editing for hemophilia was developed using comprehensive methodologies, gathering insight through qualitative research and in-depth interviews, language audits, and workshops, with input from lived experience experts, leading clinicians in hemophilia, gene therapy experts, and scientific and patient organizations. This lexicon serves as a gold standard template for future comprehensive patient lexicon development strategy and could be applied to other therapeutic areas where treatments are being developed and standardized, or where accessible vocabulary for patients, healthcare professionals, and the affected community is lacking. This communication highlights the need for lexicon development for advanced gene editing treatments across therapeutic areas to support standardized understanding and enhance communication.
Corneal neovascularization is a sight-threatening condition for which current treatments such as anti-VEGF agents are limited by invasiveness and side effects. We present the first non-viral, CRISPR/Cas9-based gene thera...Corneal neovascularization is a sight-threatening condition for which current treatments such as anti-VEGF agents are limited by invasiveness and side effects. We present the first non-viral, CRISPR/Cas9-based gene therapy delivered via topical eye drops that penetrates the cornea and inhibits pathological neovascularization. Cas9 ribonucleoproteins (RNPs) targeting the Vegfa gene were complexed with a liposomal carrier (lipofectamine) and administered to mice after alkali burn injury to the cornea. This approach achieved approximately 2% gene editing at the Vegfa locus in vivo, which significantly reduced local VEGF-A expression. Consequently, treated corneas showed markedly decreased macrophage infiltration and robust suppression of both hemangiogenesis and lymphangiogenesis compared to untreated controls. These findings demonstrate that even modest in vivo gene editing can yield a strong therapeutic effect, highlighting a clinically relevant strategy for controlling corneal angiogenesis. Our study introduces a feasible and safe topical CRISPR therapy for corneal diseases, offering a potential alternative to invasive or virus-based gene delivery methods.
Adeno-associated virus (AAV) vectors are essential tools for gene therapy (GT), yet preexisting immunity can hinder their efficacy. This study examines the seroprevalence of total binding IgG against AAV serotypes 2, 5,...Adeno-associated virus (AAV) vectors are essential tools for gene therapy (GT), yet preexisting immunity can hinder their efficacy. This study examines the seroprevalence of total binding IgG against AAV serotypes 2, 5, 6, 8, and 9, and neutralizing antibodies (NAb) against AAV 6, 8, and 9 serotypes in a healthy adult Spanish population. Using enzyme-linked immunosorbent assays (ELISA) for IgG and luciferase-based assays for NAb, we found that IgG seroprevalence was highest for AAV6 (84.4%), followed by AAV2 (74.4%) and AAV8 (60.0%), with the lowest prevalence observed for AAV9 (51.2%) and AAV5 (40.4%). For NAb, AAV6 showed the highest prevalence (62.8%), followed by AAV8 (60.4%) and AAV9 (52.4%). Co-prevalence analysis revealed high rates among all IgG-AAV serotypes tested (ranging from 46.4% to 97.0%) and significant co-occurrence of NAb against AAV6, AAV8, and AAV9 (83.0% to 91.0%). High NAb levels correlated with high IgG levels in all samples. The variability in seroprevalence across populations highlights the importance of personalized and validated testing to optimize treatment outcomes and ensure the safety of AAV-based gene therapies.
Cell and gene therapies may provide life-extending treatments for patients. However, paying for these therapies using a single upfront payment will be challenging because of uncertainty about long-term clinical effective...Cell and gene therapies may provide life-extending treatments for patients. However, paying for these therapies using a single upfront payment will be challenging because of uncertainty about long-term clinical effectiveness and affordability. Developers, recognizing the challenges of paying for these therapies, have offered payers 5-year outcomes-based installment plans. The short length of these plans, however, does little to address uncertainties about the cost-effectiveness of paying for these therapies. Instead, we propose to offer 30-year performance-based annuities that shift payments to match the expected accrual of clinical benefits more closely. Using securitization techniques combined with long-term performance-based annuities, we demonstrate that in the case of the gene therapy Zolgensma, this mechanism is effective at mitigating concerns over value and affordability for payers. In summary, our proposal for financing cell and gene therapies creates a viable incentive for developers, while also balancing long-term effectiveness and budget impact concerns from payers and access challenges for patients.
The liver is a primary target for recombinant adeno-associated viral (rAAV) vectors, yet the influence of serotype, sex, and liver zonation on transduction and transcriptomic changes remains incompletely understood. This...The liver is a primary target for recombinant adeno-associated viral (rAAV) vectors, yet the influence of serotype, sex, and liver zonation on transduction and transcriptomic changes remains incompletely understood. This proof-of-concept study employs spatial transcriptomics alongside single-nucleus RNA sequencing to map the spatial distribution and impacts of rAAV2- and rAAV9-CMV-EGFP vectors in male and female mouse livers. Spatial transcriptomics provided precise transgene mapping and highlighted that CMV-EGFP rAAV vectors deregulate hepatocellular lipid metabolism, the circadian clock, and the immune/stress response with sex specific differences. Lipid metabolism genes (Elovl3, Chka, Irs2, Ppard), were consistently deregulated across all zones of the liver lobule in male and female rAAV2- and rAAV9-CMV-EGFP-treated mice, while Srebf1, Tlcd4, Cpt2, and Acot1 exhibited sex-specific patterns. Circadian clock modulators (Dbp, Tef, Arntl, Nfil3, Nr1d1/Nr1d2) were altered independently of zonation. The study found sex-specific downregulation of immune and stress-response genes and pathways, including Gadd45g and hypoxia pathways. TGF-β and EGFR pathways were upregulated sex-independently. Spatial transcriptomics further enabled examination of transgene and rAAV entry factor co-expression, identifying known and novel factors like Rpsa, Dpp4, Sdc1, and solute carrier proteins, highlighting its role in supporting targeted screening. Our findings demonstrate spatial transcriptomics as a powerful tool in gene therapy research and reveal novel rAAV vector effects on liver biology.
Retinitis pigmentosa (RP) is an inherited retinal disease that causes progressive vision loss, ultimately leading to blindness. Currently, RP is mostly untreatable, and patients can only manage their symptoms through sup...Retinitis pigmentosa (RP) is an inherited retinal disease that causes progressive vision loss, ultimately leading to blindness. Currently, RP is mostly untreatable, and patients can only manage their symptoms through supportive measures such as visual aids and psychotherapy. Mutations in the lecithin: retinol acyltransferase (LRAT) gene, which encodes an essential protein in the visual cycle, can cause early-onset retinitis pigmentosa. In a Dutch RP patient cohort, the c.12delC mutation in the LRAT gene was identified as the most common cause of LRAT-associated RP. Gene replacement therapy is a promising treatment strategy for these patients. To test the feasibility of this approach, we used a Brown Norway rat strain with a homologous mutation to the aforementioned patient group (c.12delA) in the rat Lrat gene, and treated them with adeno-associated virus (AAV2)-based human LRAT cDNA delivery. We combined in vivo and ex vivo techniques to determine the treatment's potential. Supplementation of the LRAT gene in the subretinal space led to morphological improvements of the retina, significantly increased the electrical response to light, and enhanced functional vision compared to sham-treated controls. Our results provide strong proof-of-concept for AAV-mediated gene replacement as a potential treatment for LRAT-associated RP.
Aniridia is a rare congenital vision-loss disorder that is caused primarily by heterozygous loss-of-function variants in the PAX6 gene. There is currently no curative treatment. Gene therapy has emerged as a powerful str...Aniridia is a rare congenital vision-loss disorder that is caused primarily by heterozygous loss-of-function variants in the PAX6 gene. There is currently no curative treatment. Gene therapy has emerged as a powerful strategy for treating inherited retinal diseases. Here, we aim to establish a systemic PAX6 gene-augmentation therapy capable of addressing retinal pathologic phenotypes in a preclinical Sey mouse model of aniridia. We evaluated the intravenous delivery of FLAG-tagged PAX6 packaged in the AAV-PHP.eB capsid. Treatment was administered on postnatal day 21 to mimic the average age at diagnosis and the likely early therapeutic window in humans. The transcript levels of the virally-delivered PAX6 were 7.1% at 1 month, and 8.9% at 5 months, of Wt endogenous levels. These transcripts produced PAX6 protein in the ganglion (GCL) and inner nuclear cell layers. Importantly, in this pilot study, a significant increase in GCL thickness at 5 months post-injection was also observed. Furthermore, this structural change in the Sey retina was preceded by a significant increase in Notch1 transcription at 1-month post-injection. Thus, we demonstrated the first successful viral-mediated augmentation of PAX6 in the retina in a preclinical mouse model of aniridia, resulting in a phenotypic change at the structural and molecular levels.
Young-onset Parkinson's disease (PD), the most common autosomal recessive familial PD, is caused by gene mutations in Parkin (PRKN). These mutations result in Parkin protein loss and reduced enzymatic activity, leading t...Young-onset Parkinson's disease (PD), the most common autosomal recessive familial PD, is caused by gene mutations in Parkin (PRKN). These mutations result in Parkin protein loss and reduced enzymatic activity, leading to severe degeneration of dopamine-producing neurons in the substantia nigra pars compacta (SNpc). Adeno-associated virus (AAV) gene therapy can directly address the cause of PRKN-PD by expressing Parkin protein at levels comparable to those observed in healthy humans. AAV9 vectors with different promoters were engineered to deliver PRKN cDNA with efficient human Parkin (hParkin) expression in dopaminergic (DA) neurons as shown in PRKN-null human induced pluripotent stem cell (iPSC) derived DA neurons. Further, we show that AAV9-PRKN treatment can protect nigral DA neurons in two mouse PD models, the 6-hydroxydopamine (6-OHDA)-lesion and the α-synuclein (α-Syn) pre-formed fibrils (PFFs)-lesion model. In summary, AAV9-PRKN gene therapy demonstrates neuroprotective properties and may represent a promising approach for PRKN-PD, with potential broader applications in idiopathic PD and other neurodegenerative diseases.
Alzheimer's disease (AD) is the most common neurodegenerative disorder, yet effective preventive or therapeutic strategies remain limited. A hallmark of AD pathology is the accumulation of insoluble amyloid-β (Aβ) aggreg...Alzheimer's disease (AD) is the most common neurodegenerative disorder, yet effective preventive or therapeutic strategies remain limited. A hallmark of AD pathology is the accumulation of insoluble amyloid-β (Aβ) aggregates, which are targeted by recent antibody-based therapies. Conversely, soluble amyloid precursor protein-alpha (sAPPα), a non-amyloidogenic cleavage product of APP, possesses neuroprotective, neurotrophic, and synaptogenic properties, and the ability to enhance memory. This study evaluated the therapeutic efficacy of adeno-associated virus variant PHP.eB (AAV-PHP.eB) encoding human sAPPα in the APPswe/PS1dE9 transgenic mouse model of AD. Six-month-old female wild-type and transgenic mice received a single intravenous injection via the tail vein. Three months post-injection, brain tissue was harvested for electrophysiological and histological analyses. The treatment significantly increased cortical sAPPα levels and fully restored hippocampal long-term potentiation (LTP) in transgenic mice. Post-mortem analyses revealed a substantial reduction in amyloid plaque burden in both the hippocampus and cortex, with minimal plaque progression from the time of injection. However, no significant changes were observed in astrocytic (GFAP) or microglial (Iba-1) coverage, nor in soluble and insoluble Aβ1-40 or Aβ1-42 levels. These findings suggest that systemic AAV-PHP.eB-mediated sAPPα delivery can ameliorate synaptic dysfunction and aggregated amyloid pathology in AD, highlighting its potential as a therapeutic strategy.
Thoracic aortic disease poses a significant threat due to its high mortality rates and genetic. underpinnings. While gene therapy holds promise for cures, the challenge lies in achieving. effective and safe gene delivery...Thoracic aortic disease poses a significant threat due to its high mortality rates and genetic. underpinnings. While gene therapy holds promise for cures, the challenge lies in achieving. effective and safe gene delivery to the thoracic aorta. Tail vein injection (TI), is hindered by. off-target effects and hepatotoxicity, and traditional blinded percutaneous left heart injection. (TLI) carries a heightened risk of bleeding and mortality. To address these limitations. ultrasound-guided techniques present a viable solution. Adeno-associated virus (AAV) vectors. were delivered into the thoracic aorta of mice through TI, TLI, and ultrasound-guided. percutaneous left heart injection(ULI). While all three injection methods can achieve gene transduction in the thoracic aorta, the ULI approach provides the optimal balance between. high transduction efficiency and safety. The ULI method represents an efficient and safe. strategy for targeted gene delivery to the mouse thoracic aorta, providing a powerful tool for preclinical aortic gene therapy research that warrants broader application.
Blood stem cell gene therapy to treat hemoglobinopathies is beginning to transform health for small numbers of patients in the U.S. and Europe, where these conditions qualify as rare diseases. Yet hemoglobinopathies are...Blood stem cell gene therapy to treat hemoglobinopathies is beginning to transform health for small numbers of patients in the U.S. and Europe, where these conditions qualify as rare diseases. Yet hemoglobinopathies are common globally, disproportionately affecting low- and middle-income countries (LMICs), creating an ethical imperative to ensure access where disease burden is greatest. Gene therapy could have blockbuster drug potential if distributable to these regions, but cost is a major barrier. Cost-effectiveness analysis (CEA) models are seldom adapted to low-income settings, where limited data and resources constrain efforts to contextualize high-income evidence. Here, we present a novel framework to evaluate high-income country authorized gene therapies in LMIC contexts. Uganda, where sickle cell disease (SCD) imposes a major burden and no curative therapies are available, is the test case. We evaluate cost-effectiveness of gene therapy for adolescents and adults with SCD in Uganda, adapting U.S. evidence to local economic conditions. Using a three-state Markov model to estimate lifetime costs of standard-of-care in Uganda, two U.S.-based CEA models were adapted using scaling factors and applied to two authorized gene therapies for SCD, Lyfgenia™ (lovo-cel) and Casgevy® (exa-cel), assuming biologically consistent efficacy across populations. Incremental cost-effectiveness ratios (ICERs) were calculated from healthcare and societal perspectives, with internationally accepted gross domestic product-based thresholds. This study demonstrates that Casgevy could be cost-effective in Uganda at a scaled cost when societal benefits are considered. This framework enables CEAs for emerging therapies where local clinical trial data are limited, supporting local decision-makers, global funders, and manufacturers in advancing equitable access to transformative therapies in LMICs.
The advancement of adeno-associated virus (AAV) gene therapy applications faces significant challenges, particularly in the separation of empty (E), full (F), and intermediate capsids during manufacturing, as well as sca...The advancement of adeno-associated virus (AAV) gene therapy applications faces significant challenges, particularly in the separation of empty (E), full (F), and intermediate capsids during manufacturing, as well as scaling up downstream processing. This study demonstrates the effectiveness of monolithic chromatography in maintaining high flow rates without sacrificing resolution across scales, making it ideal for large-scale applications. The CIMmultus® HR columns ensure high reproducibility at various purification scales, achieving ± 3% isoconductivity elution of empty AAV2/8 capsids.In combination with the improved method, it enriches full AAV capsids to 88% ± 9% with genome recovery of 77% ± 3% within this study. The screening of polishing columns exhibited highest enrichment and recovery of full capsids with an improved method developed on QA HR preparative column. High-throughput screening (HTS) on CIM® QA HR Monolithic Well Plate and subsequent single-column experiments confirmed these findings, facilitating predictions for implementing step elution approaches. The high reproducibility of the columns enabled the development of step elution methods in both bind-elute and flow through modes, with both approaches yielding comparable recovery and purity of full AAV capsids. Ultimately, the optimized AAV polishing method was successfully scaled up from 1 to 8000 mL monolith using step elution approach.
Patients diagnosed with esophageal cancer (EC) currently rely on treatments given at specialist care centers (surgery, chemotherapy, radiation), which despite their low cure rates are extremely life-disruptive, cause sev...Patients diagnosed with esophageal cancer (EC) currently rely on treatments given at specialist care centers (surgery, chemotherapy, radiation), which despite their low cure rates are extremely life-disruptive, cause severe pain, and have strong side effects. In particular, dysphagia is one of the most distressing and debilitating symptoms in patients with cancer-related esophageal obstruction. There is clearly an urgent need for new effective and accessible therapies for EC patients that allow patients to continue normal activity as much as possible. Here, we describe a drinkable methylcellulose/xanthan gum-based gene therapy foam that coats the esophagus and accumulates an apoptosis-inducing gene therapy drug (mRNA lipid nanoparticles encoding Pseudomonas exotoxin A) at the tumorous esophageal stricture. In an in vitro EC tissue model, we show that gene therapy foam induces 110-fold higher tumor regression compared to suspension treatment. We also establish that gene therapy foam given prior to radiotherapy strongly enhances anti-tumor effects. Once implemented in the clinic, this treatment, which can be administered orally by a local family doctor or at home by the patient or caregiver, could maximize the time EC patients can live normal lives outside of the hospital and allow them to maintain their ability to swallow and eat.
Neovascular age-related macular degeneration (nAMD) is a major cause of irreversible vision loss in the elderly, driven by choroidal neovascularization and dysregulated vascular endothelial growth factor (VEGF) signaling...Neovascular age-related macular degeneration (nAMD) is a major cause of irreversible vision loss in the elderly, driven by choroidal neovascularization and dysregulated vascular endothelial growth factor (VEGF) signaling. While anti-VEGF injections have transformed management, their frequent administration imposes a substantial burden on patients and limits adherence. Adeno-associated virus (AAV)-based gene therapy offers sustained intraocular delivery of anti-angiogenic agents with a single treatment, potentially overcoming these limitations. This review summarizes the rationale for AAV use in ocular gene therapy, compares major delivery routes, and highlights leading clinical candidates, including RGX-314, ADVM-022, 4D-150, and NG101. Advances in vector engineering, promoter optimization, and immune modulation are discussed alongside key challenges such as preexisting immunity and inflammation. Future directions include next-generation capsids, combination regimens, and precision patient selection. Collectively, these developments position AAV-based gene therapy as a promising strategy to redefine the therapeutic landscape of nAMD.
Lai KY, Nie S, Chen YA
… +14 more, Tiambeng TN, Tang S, Huang Y, Yan Y, Mishra S, Al-Rubaye H, Hermann A, Liu N, Rosconi M, Li N, Shameem M, Wang S, Zhi L, Liu D
While recombinant adeno-associated virus (AAV) holds significant promise for effective and durable gene delivery for gene therapy, a thorough understanding of the critical quality attributes (CQAs) along with the degrada...While recombinant adeno-associated virus (AAV) holds significant promise for effective and durable gene delivery for gene therapy, a thorough understanding of the critical quality attributes (CQAs) along with the degradation pathways of AAV under the various stresses that may occur during manufacturing, storage, and handling remains limited. To address this gap, we performed a comprehensive forced degradation study to elucidate the degradation pathways of AAV8 under a series of stress conditions, such as oxidation, extreme pH, high temperature, freeze-thaw, and agitation. Our results show that, under these stress conditions, distinct post-translational modifications (PTM), including methionine oxidation, asparagine deamidation, and aspartic acid isomerization, along with multiple physical degradation pathways, including capsid aggregation, viral protein fragmentation, and genome DNA leakage, could occur. Alterations in AAV8 biological activity were frequently attributed to the combination effect from chemical and physical degradation mechanisms. The results from this study provide a valuable insight into the establishment of stability-indicating methods and the identification of CQAs for AAV. It will also support the development of robust manufacturing process as well as stable and efficacious AAV gene therapy drug products.
Previously, we confirmed that BaEV-LVs outperformed VSV-G-LVs for gene delivery or correction of human T cells, B cells, NK cells and HSPCs correlating with high expression of its receptors, ASCT-1 and ASCT-2 on these ce...Previously, we confirmed that BaEV-LVs outperformed VSV-G-LVs for gene delivery or correction of human T cells, B cells, NK cells and HSPCs correlating with high expression of its receptors, ASCT-1 and ASCT-2 on these cells. Since HERV-W gp uses the same entry receptors, we compared transduction efficiencies for BaEV-LVs and HERV-W-LVs in hematopoietic cells. HERV-W LV transduction was efficient but inferior to BaEV-LV in TCR-stimulated T cells (40% versus 80%) and this low efficiency was even more pronounced in IL-7/IL-15 pre-stimulated T cells. BaEV-LVs were significantly superior over HERV-W-LVs for the transduction of B cells and NK cells. High HERV-W-LV mediated transduction levels were achieved for pre-stimulated hCD34+ cells, which remained though lower than for the BaEV-LVs. Additionally, BaEV-LVs reached over 80% of transduction in severe combined immunodeficiency (SCID) repopulating cells (SRC) in 6/6 engrafted NBSGW mice. HERV-W-LVs reached this transduction level in 1/5 mice, while 3/5 engrafted NBSGW mice reached significantly lower transduction levels (20-50%). For both vectors the transduction levels were equivalent in the lymphoid and myeloid lineages in all hematopoietic tissues, suggesting transduction of immature HSPCs. Summarizing, BaEV-LVs outperformed HERV-W-LVs for transduction of important gene therapy target cells such as NK, B, T cells and CD34+ HSPCs.
Gottimukkala KSV, Lane DD, Cunningham R
… +10 more, Malik HS, Jwa Y, Cassidy ME, Castelli JMP, Enstrom MR, Poljakov K, Gastelum G, Ho SH, Tassa C, Adair JE
Efficient delivery of CRISPR ribonucleoproteins into primary hematopoietic stem and progenitor cells (HSPCs) is essential for durable gene editing therapies but remains challenging. Here, we advance a modular, benchtop-a...Efficient delivery of CRISPR ribonucleoproteins into primary hematopoietic stem and progenitor cells (HSPCs) is essential for durable gene editing therapies but remains challenging. Here, we advance a modular, benchtop-assembled gold-polymer hybrid nanoparticle (CRISPR-AuNP) platform that enables non-viral delivery of multiple CRISPR systems into HSPCs. Guided by a mechanistic understanding of Cas9's interaction with gold surfaces, we engineered the formulation by conjugating pre-formed RNP-polymer complexes, assembled using thiolated polyethyleneimine-polyethylene glycol, to gold nanoparticles. This system achieved efficient editing in primary CD34+ HSPCs for Cas9, Cas12a, and Cas12a-M29-1 without compromising cell viability. Notably, the nanoformulation can be assembled in under 2 h in a PCR tube for less than $70/million HSPCs treated. This work establishes a scalable, cost-effective, and accessible gene editing system with the potential to democratize CRISPR applications in HSPC research and therapy.