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At least 19 recordsLinked to original sources

PubMed2011

A versatile adeno-associated virus vector producer cell line method for scalable vector production of different serotypes.

Application of adeno-associated virus (AAV) vector in large animal studies and clinical trials often requires high-titer and high-potency vectors. A number of currently used vector production methods, based on either transient transfection or helper virus infection of cell lines, have their advantages and limitations. We previously developed a 293-cell-based producer cell line method for high-titer and high-potency AAV2 vectors. Similar to several other methods, however, it requires multiple cloning steps for the vector and packaging plasmids and a two-step transfection and selection for stable cell lines. Here we report a simplified method with several key improvements and advantages: (1) a one-step cloning of AAV vector cassette into the serotype-specific packaging plasmid; (2) a single plasmid transfection and selection for stable AAV vector producer cell lines; (3) high vector yields of different serotypes, e.g., AAV2, 8, and 9, upon infection with an E1A/E1B-deleted helper adenovirus; (4) efficient packaging of both single-stranded and double-stranded (self-complementary) AAV vectors; and (5) efficient packaging of large AAV cassettes such as a mini-dystrophin vector (5.0 kb). All cell lines were stable with growth rates identical to the parental 293 cells. The vector yields were consistent among serotypes, with 5 × 10(13) to 8 × 10(13) vector genome particles per Nunc cell factory (equivalent to 40 15-cm plates). The vectors showed high potency for in vitro and in vivo transduction. In conclusion, the simple and versatile AAV producer cell line method can be useful for large scale AAV vector production in preclinical and clinical studies.

Biotechnology↗

PubMed2009

Reducing the risk of adeno-associated virus (AAV) vector mobilization with AAV type 5 vectors.

Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to "mobilization" of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.

Animals↗

PubMed2025

AAV6 vectors provide superior gene transfer compared to AAV9 vectors following intramyocardial administration.

Cardiac gene therapy using adeno-associated viral (AAV) vectors holds great promise for treating heart diseases but would benefit from more potent AAV vectors. Vectors based on the AAV serotypes 6 and 9 have been used in pre-clinical gene therapy studies, yet the therapeutic outcomes varied depending on the experimental model and delivery route used. Here, we evaluated the transduction efficiency of AAV6, AAV9, and AAV9-derived MyoAAVs for local cardiac delivery. Vectors were tested in neonatal rat ventricular myocytes, and subsequently in mouse hearts by direct intramyocardial injection. Vector genome levels, mRNA expression levels, and fluorescence were measured. The AAV6 and AAV9 vectors were further validated in porcine hearts, human-induced pluripotent stem-cell-derived cardiomyocytes, and human atrial myocardial slices. In both rat cardiomyocytes and mouse hearts, AAV6 exhibited the highest transduction efficiency. Direct comparison of the AAV6 and AAV9 vectors in porcine and human models confirmed that AAV6 is more potent. In conclusion, AAV6 vectors are superior to AAV9 and its derivative vectors for cardiac transduction by direct intramyocardial injection. In addition, the in vivo transduction efficiency correlates with in vitro and ex vivo assays, thereby facilitating the development of more potent AAV variants for cardio-selective delivery methods.

AAV vector↗

PubMed2026

Evaluation of transduction properties and vaccine efficacy of a simian adenovirus type 25-based vector.

Although human adenovirus serotype 5 (Ad5) is widely used as a vaccine vector for infectious diseases due to its high transduction efficiency, pre-existing immunity to Ad5 in many people reduces vaccine efficacy. To address this limitation, simian Ad vectors, such as ChAdOx1 and ChAdOx2, have been explored as alternative vaccine platforms. ChAdOx2 is based on simian Ad25 (SAd25), but the fundamental characteristics of gene transduction by SAd25-based vectors have not been fully elucidated. This study aimed to characterize the gene transduction efficiency, tissue distribution, and immunogenicity of an SAd25-based vector in comparison with those of the Ad5 vector following various routes of administration. Compared with intravenous administration of the Ad5 vector, intravenous administration of the SAd25 vector showed distinct biodistribution patterns, including reduced liver accumulation and predominant expression in the lung. Transduction by the SAd25 vector was not inhibited by human serum, whereas transduction by the Ad5 vector was inhibited, indicating that the SAd25 vector, but not the Ad5 vector, can evade pre-existing Ad immunity. Although intramuscular administration of the SAd25 vector induced lower transgene product-specific antibody production than intramuscular administration of the Ad5 vector, gene expression and Ad genome distribution mediated by the SAd25 vector, but not the Ad5 vector, were localized only to the muscle at the administration site. Intranasal administration of the SAd25 vector induced an antigen-specific antibody response in serum more rapidly than intranasal administration of the Ad5 vector. The SAd25 vector induced antigen-specific antibody production in bronchoalveolar lavage fluid (BALF) that was comparable to that induced by the Ad5 vector. These findings provide essential insights into the biological characteristics of the SAd25 vector, supporting its potential as a safe and effective vaccine vector.

Animals↗

PubMed2010

AAV's anatomy: roadmap for optimizing vectors for translational success.

Adeno-Associated Virus based vectors (rAAV) are advantageous for human gene therapy due to low inflammatory responses, lack of toxicity, natural persistence, and ability to transencapsidate the genome allowing large variations in vector biology and tropism. Over sixty clinical trials have been conducted using rAAV serotype 2 for gene delivery with a number demonstrating success in immunoprivileged sites, including the retina and the CNS. Furthermore, an increasing number of trials have been initiated utilizing other serotypes of AAV to exploit vector tropism, trafficking, and expression efficiency. While these trials have demonstrated success in safety with emerging success in clinical outcomes, one benefit has been identification of issues associated with vector administration in humans (e.g. the role of pre-existing antibody responses, loss of transgene expression in non-immunoprivileged sites, and low transgene expression levels). For these reasons, several strategies are being used to optimize rAAV vectors, ranging from addition of exogenous agents for immune evasion to optimization of the transgene cassette for enhanced therapeutic output. By far, the vast majority of approaches have focused on genetic manipulation of the viral capsid. These methods include rational mutagenesis, engineering of targeting peptides, generation of chimeric particles, library and directed evolution approaches, as well as immune evasion modifications. Overall, these modifications have created a new repertoire of AAV vectors with improved targeting, transgene expression, and immune evasion. Continued work in these areas should synergize strategies to improve capsids and transgene cassettes that will eventually lead to optimized vectors ideally suited for translational success.

Cystic Fibrosis↗

PubMed2026

Development of receptor and oncogene-responsive luciferase reporter vectors that are activated via kinase signaling to AP-1.

This study aimed to generate lentiviral vectors carrying an array of AP-1 motifs driving luciferase gene expression as reporters of mitogen-activated protein kinase (MAPK) activity. We created a series of vectors based on LeGO-iG that were used to generate stably transduced leukemia cell lines. A vector termed LEGO-AP1×6-GM55 containing an array of 6 AP-1 sites linked to the minimal CSF2 promoter was sufficient to support high levels of MAPK-inducible luciferase activity in leukemic cell lines that was suppressed by MAPK inhibitors. The inclusion of a putative chromatin priming element encompassing RUNX and ETS motifs increased the activity of these vectors. The additional inclusion of the full-length mouse CSF2 promoter, or the human DUSP5 promoter further increased the MAPK-dependent activity of these vectors in leukemic cells. These vectors support moderate levels of constitutive activity in cells carrying mutations that activate the RAS/RAF/MEK MAPK signaling pathway, and high-level activity after direct activation of MAPK signaling. They also respond to T-cell receptor activation via MAPK and Ca2+ signaling pathways. This resource will now make it easier to track receptor or oncogene-inducible MAPK activity in cultured cells, and potentially in tumors, close to real-time.

Humans↗

PubMed2024

Enhanced transgene expression from single-stranded AAV vectors in human cells in vitro and in murine hepatocytes in vivo.

We identified that distal 10 nucleotides in the D-sequence in AAV2 inverted terminal repeat (ITR) share partial sequence homology to 1/2 binding site of glucocorticoid receptor-binding element (GRE). Here, we describe that (1) purified GR binds to AAV2 D-sequence, and the D-sequence competes with GR binding to its cognate binding site; (2) dexamethasone-mediated activation of GR pathway significantly increases the transduction efficiency of AAV2 vectors in human cells; (3) human osteosarcoma cells, U2OS, which lack expression of GR, are poorly transduced by AAV2 vectors, but stable transfection with a GR expression plasmid restores vector-mediated transgene expression; (4) replacement of the distal 10 nucleotides in the D-sequence of the AAV2 ITR with a full-length GRE consensus sequence significantly enhances transgene expression in human cells in vitro and in murine hepatocytes in vivo; and (5) none of the ITRs in AAV1, AAV3, AAV4, AAV5, and AAV6 genomes contains the GRE 1/2 binding site, and insertion of a full-length GRE consensus sequence in the AAV6-ITR also significantly enhances transgene expression from AAV6 vectors, both in vitro and in vivo. These novel vectors, termed generation Y AAV vectors, which are serotype, transgene, or promoter agnostic, should be useful in human gene therapy.

AAV vectors↗

PubMed2022

AAV vectors: The Rubik's cube of human gene therapy.

Defective genes account for ∼80% of the total of more than 7,000 diseases known to date. Gene therapy brings the promise of a one-time treatment option that will fix the errors in patient genetic coding. Recombinant viruses are highly efficient vehicles for in vivo gene delivery. Adeno-associated virus (AAV) vectors offer unique advantages, such as tissue tropism, specificity in transduction, eliciting of a relatively low immune responses, no incorporation into the host chromosome, and long-lasting delivered gene expression, making them the most popular viral gene delivery system in clinical trials, with three AAV-based gene therapy drugs already approved by the US Food and Drug Administration (FDA) or European Medicines Agency (EMA). Despite the success of AAV vectors, their usage in particular scenarios is still limited due to remaining challenges, such as poor transduction efficiency in certain tissues, low organ specificity, pre-existing humoral immunity to AAV capsids, and vector dose-dependent toxicity in patients. In the present review, we address the different approaches to improve AAV vectors for gene therapy with a focus on AAV capsid selection and engineering, strategies to overcome anti-AAV immune response, and vector genome design, ending with a glimpse at vector production methods and the current state of recombinant AAV (rAAV) at the clinical level.

United States↗

PubMed2011

Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector.

Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer.

Amino Acid Sequence↗

PubMed2026

Astigmatic vector outcomes after FS-LASIK versus SMILE for high myopic astigmatism: a single-center retrospective comparative cohort study without cyclotorsion compensation.

PURPOSE: To compare astigmatic correction vector outcomes between femtosecond laser-assisted in situ keratomileusis (FS-LASIK) and small-incision lenticule extraction (SMILE, also termed Keratorefractive Lenticule Extraction, KLEx) without intraoperative cyclotorsion compensation in patients with high myopic astigmatism (- 2.00 to - 3.75 D), and to clarify procedure-specific correction tendencies under this non-standardized alignment protocol. METHODS: This single-center retrospective comparative cohort study enrolled 155 eyes (one eye randomly selected per patient) that underwent FS-LASIK (80 eyes) or SMILE/KLEx (75 eyes) for high myopic astigmatism correction from January 2023 to July 2024 in Beijing Fenglian Jiayue Lige Clinic. Intraoperative cyclotorsion compensation was intentionally disabled to isolate inherent procedural astigmatism correction characteristics. Standardized Alpins vectorial analysis was performed at 3 months and 12 months postoperatively. PRIMARY ENDPOINT: 12-month Alpins correction index (CI). Multivariable propensity score adjustment was applied to mitigate confounding by clinical treatment selection bias. Statistical multiplicity control was implemented for secondary vector and visual outcomes. RESULTS: Baseline demographic, refractive, corneal and ocular biometric parameters were balanced between groups after propensity matching. No statistically significant intergroup differences were detected in uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), residual cylinder, safety index or efficacy index at 3 and 12 months (all P > 0.05). Under the non-cyclotorsion-compensated protocol, significant intergroup differences were identified in the magnitude of surgically induced astigmatism (SIA), correction index (CI), and magnitude error (ME) at both follow-up timepoints (all P < 0.0001). Target induced astigmatism (TIA), difference vector (DV), index of success (IOS), and angle error (AE) magnitudes were comparable between groups (all P > 0.05). The vector mean axis of DV differed significantly between groups at 3 and 12 months (Watson-Williams circular test, all P < 0.0001). No reoperations were documented in clinic medical records for either cohort. No standardized dry eye questionnaires, tear film testing or corneal nerve density metrics were collected to quantify dry eye adverse events; only unstructured clinical notes were reviewed for complication screening. CONCLUSIONS: Under surgical alignment without cyclotorsion compensation, FS-LASIK and SMILE/KLEx both yielded acceptable visual and refractive safety/efficacy for high myopic astigmatism (- 2.00 to - 3.75 D) at 1-year follow-up, but demonstrated divergent astigmatism correction tendencies: FS-LASIK exhibited relative astigmatism overcorrection (vector mean DV: - 0.35 ± 0.43 D × 91°, CI > 1), while SMILE/KLEx showed relative undercorrection (vector mean DV: - 0.21 ± 0.53 D × 12°, CI < 1). These correction biases are specific to the study's manual limbal alignment protocol without cyclotorsion tracking and cannot be generalized to modern optimized surgical platforms equipped with automated cyclotorsion compensation. Residual refractive errors across both groups are likely multifactorial, including differential corneal stromal healing responses, divergent femtosecond/excimer laser tissue modification mechanisms, and uncorrected intraoperative ocular cyclotorsion.

Humans↗

PubMed2016

Adeno-associated virus (AAV) vectors in cancer gene therapy.

Gene delivery vectors based on adeno-associated virus (AAV) have been utilized in a large number of gene therapy clinical trials, which have demonstrated their strong safety profile and increasingly their therapeutic efficacy for treating monogenic diseases. For cancer applications, AAV vectors have been harnessed for delivery of an extensive repertoire of transgenes to preclinical models and, more recently, clinical trials involving certain cancers. This review describes the applications of AAV vectors to cancer models and presents developments in vector engineering and payload design aimed at tailoring AAV vectors for transduction and treatment of cancer cells. We also discuss the current status of AAV clinical development in oncology and future directions for AAV in this field.

Capsid Proteins↗

PubMed2026

Phytoplasma-plant interactions: effector-mediated host reprogramming, hormonal crosstalk, metabolic alterations and plant-mediated vector manipulation.

Phytoplasmas are wall-less, phloem-restricted bacterial pathogens that infect over 1,000 plant species, causing substantial losses in agriculture, horticulture, and forestry worldwide. Despite their reduced genomes and limited metabolic autonomy, these obligate parasites colonize diverse hosts through secreted effector proteins that extensively reprogram plant development, metabolism, immune signalling, and vector interactions. Advances in genomics, transcriptomics, proteomics, metabolomics, and functional studies have substantially clarified the molecular basis of phytoplasma pathogenicity and symptom development. This review synthesizes current understanding of phytoplasma-plant interactions, covering phytoplasma biology, genome evolution, and the infection cycle across plant and insect vector hosts. We examine the molecular functions of key effectors, SAP11, SAP54/PHYL1, SAP05, TENGU, SWP1, and recently identified virulence factors, focusing on how they target host transcription factors, phytohormone networks, protein degradation pathways, and immune responses to promote colonization and disease progression. We further discuss how phytoplasma infection disrupts phytohormone signalling, primary and secondary metabolism, and developmental programs to produce characteristic disease symptoms, with particular attention to pathogen-induced changes in host volatiles and nutritional quality that alter vector behaviour and enhance transmission. Finally, we summarize insights from multi-omics studies and emerging management strategies, including CRISPR-based genome editing, RNAi, rapid molecular diagnostics, resistant cultivars, microbiome-based approaches, and sustainable vector control, and highlight key knowledge gaps and priorities for developing effective, environmentally sustainable phytoplasma disease management.

Phytoplasma↗

PubMed2024

Shedding light on Klebsiella pneumoniae virulence: Engineering of broad host range bioluminescence reporter vectors for transcriptional analysis in drug resistant pathogens.

In this work, we report the construction of four bacterial luciferase-based promoter probe vectors with an expanded set of selectable markers, designed to facilitate their use in antibiotic-resistant bacteria. These vectors contain the low-copy-number, broad-host-range pBBR origin of replication and an origin of transfer, allowing efficient conjugative transformation into various bacterial genera. The broad host range origin also enables their use in bacterial strains that harbor other plasmids, as the pBBR origin is compatible with a wide variety of other plasmid replication systems. The utility of these vectors was demonstrated by quantifying capsule gene expression in both classical and hypervirulent Klebsiella pneumoniae strains lacking tolC, which encodes the outer membrane pore protein for tripartite transport systems. Our results revealed that the tolC mutation reduced capsule gene expression, highlighting a critical role for tolC in K. pneumoniae pathobiology and the utility of bioluminescence for studying gene expression in real time. These new vectors provide a flexible platform for circumventing antibiotic resistance phenotypes and studying gene expression across diverse bacterial species, including strains containing additional plasmids.

Klebsiella pneumoniae↗

PubMed2025

Quantifying the mutational landscape of retroviral and lentiviral vectors in gene therapy patients.

Adenosine deaminase severe combined immunodeficiency (ADA-SCID) is a monogenic disorder caused by mutations in the ADA gene. Gene therapy using γ-retroviral and lentiviral vector gene addition approaches have shown curative results. We sequenced the ADA transgene in transduced CD3+ T cells, and in peripheral blood cells from patients treated with autologous CD34+ cells transduced with either a γ-retroviral or lentiviral ADA gene vector to assess transgene mutational profiles. In both CD3+ T cells and ADA-SCID patients' cells treated with the lentiviral vector, we observed significantly higher occurrences of guanine (G)-to-adenosine (A) base substitutions than with the γ-retroviral vector. We hypothesized that this G-to-A mutational signature was due to the APOBEC3 cytosine deaminase protein family. By knocking out APOBEC3 genes in HEK239T packaging cells, APOBEC3-mediated mutagenesis decreased by 91.2% along the transgene in CD34+ transduced cells in comparison to CD34+ cells transduced with lentiviral supernatant packaged in parental HEK293T cells.

Humans↗

PubMed2026

Transforming Plant Viruses into Vectors for Next-Generation Agriculture-A Review.

Plant viral vectors have evolved from tools for transient gene expression into a versatile platform for precise genetic intervention, offering a rapid, transgene-free alternative to conventional crop transformation. This review critically assesses their engineering for scalable field application, moving beyond foundational techniques like virus-induced gene silencing (VIGS). We highlight how advanced vector design, including deconstructed genomes and synthetic regulatory circuits enhances cargo capacity, specificity, and biosafety. The integration of viral delivery with CRISPR-Cas systems has unlocked virus-induced genome editing (VIGE), base editing, and prime editing, enabling heritable trait modification without tissue culture. However, the transition from proof-of-concept in model plants to robust field technology hinges on overcoming critical bottlenecks: expanding host range through chimeric vectors, ensuring environmental containment, and developing scalable delivery methods such as nano-formulations or adjusted agroinfiltration protocols. We evaluate these delivery routes and emerging synergies with nanobiotechnology for targeted and efficient applications. While challenges in regulation, public perception, and large-scale production persist, the strategic engineering of viral vectors for stability, specificity, and safety positions them as a transformative, next-generation biotechnological input for achieving sustainable crop improvement and protection under changing climatic conditions.

Plant Viruses↗

PubMed2015

In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector.

Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina.

Dependovirus↗

PubMed2023

Evolution and assembly of Anopheles aquasalis's immune genes: primary malaria vector of coastal Central and South America and the Caribbean Islands.

Anophelines are vectors of malaria, the deadliest disease worldwide transmitted by mosquitoes. The availability of genomic data from various Anopheles species allowed evolutionary comparisons of the immune response genes in search of alternative vector control of the malarial parasites. Now, with the Anopheles aquasalis genome, it was possible to obtain more information about the evolution of the immune response genes. Anopheles aquasalis has 278 immune genes in 24 families or groups. Comparatively, the American anophelines possess fewer genes than Anopheles gambiae s. s., the most dangerous African vector. The most remarkable differences were found in the pathogen recognition and modulation families like FREPs, CLIP and C-type lectins. Even so, genes related to the modulation of the expression of effectors in response to pathogens and gene families that control the production of reactive oxygen species were more conserved. Overall, the results show a variable pattern of evolution in the immune response genes in the anopheline species. Environmental factors, such as exposure to different pathogens and differences in the microbiota composition, could shape the expression of this group of genes. The results presented here will contribute to a better knowledge of the Neotropical vector and open opportunities for malaria control in the endemic-affected areas of the New World.

Animals↗

PubMed2025

Borrelia burgdorferi loses essential genetic elements and cell proliferative potential during stationary phase in culture but not in the tick vector.

The Lyme disease agent Borrelia burgdorferi is a polyploid bacterium with a segmented genome in which both the chromosome and over 20 distinct plasmids are present in multiple copies per cell. This pathogen can survive for at least 9 months in its tick vector in an apparent dormant state between blood meals, without losing cell proliferative capability when re-exposed to nutrients. Cultivated B. burgdorferi cells grown to stationary phase or resuspended in nutrient-limited media are often used to study the effects of nutrient deprivation. However, a thorough assessment of the spirochete's ability to recover from nutrient depletion has been lacking. Our study shows that starved B. burgdorferi cultures rapidly lose cell proliferative ability. Loss of genetic elements essential for cell proliferation contributes to the observed proliferative defect in stationary phase. The gradual decline in copies of genetic elements is not perfectly synchronized between chromosomes and plasmids, generating cells that harbor one or more copies of the essential chromosome but lack all copies of one or more non-essential plasmids. This phenomenon likely contributes to the well-documented issue of plasmid loss during in vitro cultivation of B. burgdorferi. In contrast, B. burgdorferi cells from ticks starved for 14 months showed no evidence of reduced cell proliferative ability or plasmid loss. Beyond their practical implications for studying B. burgdorferi, these findings suggest that the midgut of the tick vector offers a unique environment that supports the maintenance of B. burgdorferi's segmented genome and cell proliferative potential during periods of tick fasting.IMPORTANCEBorrelia burgdorferi causes Lyme disease, a prevalent tick-borne illness. B. burgdorferi must survive long periods (months to a year) of apparent dormancy in the midgut of the tick vector between blood meals. Resilience to starvation is a common trait among bacteria. However, this study reveals that, in laboratory cultures, B. burgdorferi poorly endures starvation and rapidly loses viability. This decline is linked to a gradual loss of genetic elements required for cell proliferation. These results suggest that the persistence of B. burgdorferi in nature is likely shaped more by unique environmental conditions in the midgut of the tick vector than by an innate ability of this bacterium to endure nutrient deprivation.

Borrelia burgdorferi↗