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An episome is a special type of plasmid, which remains as a part of the eukaryotic genome without integration. Episomes manage this by replicating together with the rest of the genome and subsequently associating with metaphase chromosomes during mitosis. Episomes do not degrade, unlike standard plasmids, and can be designed so that they are not epigenetically silenced inside the eukaryotic cell nucleus.[1] Episomes can be observed in nature in certain types of long-term infection by adeno-associated virus or Epstein-Barr virus. In 2004, it was proposed that non-viral episomes might be used in genetic therapy for long-term change in gene expression.[2]

As of 1999, there were many known sequences of DNA (deoxyribonucleic acid) that allow a standard plasmid to become episomally retained. One example is the S/MAR sequence.[3]

The length of episomal retention is fairly variable between different genetic constructs and there are many known features in the sequence of an episome which will affect the length and stability of genetic expression of the carried transgene. Among these features is the number of CpG sites which contribute to epigenetic silencing of the transgene carried by the episome.[4]

Mechanism of episomal retention

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The mechanism behind episomal retention in the case of S/MAR episomes is generally still uncertain. As of 1985, in the case of latent Epstein-Barr virus infection, episomes seemed to be associated with nuclear proteins of the host cell through a set of viral proteins.[5]

Episomes in prokaryotes

[edit]

Episomes in prokaryotes are special sequences which can divide either separate from or integrated into the prokaryotic chromosome.[6]

References

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Episome

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An episome is a genetic element, typically a circular DNA molecule, that can exist and replicate autonomously in the cytoplasm of a host cell or integrate into the host chromosome and replicate as part of it. This dual capability distinguishes episomes from standard plasmids, which replicate independently but do not integrate into the genome. The term was coined in 1958 by François Jacob and Élie L. Wollman to describe extrachromosomal elements in bacteria capable of such behavior. In , episomes were first identified through studies of genetic transfer mechanisms, such as conjugation. A classic example is the F (fertility) plasmid in , which exists as an autonomous replicon in F⁺ strains but can integrate into the bacterial chromosome to form high-frequency recombination (Hfr) strains, enabling the transfer of chromosomal genes during conjugation. Temperate bacteriophages, like , also behave as episomes when integrated as prophages, excising to replicate independently during the . These elements often carry genes conferring advantages, such as antibiotic resistance or metabolic capabilities, and their mobility contributes to bacterial evolution and adaptation. The concept of episomes has extended beyond prokaryotes to eukaryotes, where they typically refer to extrachromosomal, autonomously replicating DNA derived from plasmids or viruses that persist without genomic integration. In human cells, the Epstein-Barr virus (EBV) maintains its genome as a stable episome in latently infected B lymphocytes, interacting with host to regulate viral gene expression and evade immune detection. Similarly, (KSHV) forms episomes that reorganize nuclear loops to support viral persistence and oncogenesis. Synthetic episomal vectors, engineered with elements like scaffold/matrix attachment regions (S/MAR) or viral origins of replication, are used in to achieve long-term expression without risking . Recent advances as of 2025 include screening methods using AAV episomes (CrAAVe-seq) and nonviral episomal vectors for treating β-hemoglobinopathies.

Definition and Characteristics

Definition

An episome is defined as a type of molecule capable of autonomous replication within the host cell, functioning similarly to a , while also possessing the ability to undergo reversible integration into the host . This dual functionality distinguishes episomes from other genetic elements, enabling them to alternate between an independent, extrachromosomal form and a stably incorporated chromosomal state. In contrast to general plasmids, which maintain their extrachromosomal status and replicate independently without integrating into the host genome, episomes are specifically characterized by this reversible integration capability. This property allows episomes to persist in host cells through either mechanism, providing flexibility in genetic maintenance and expression. Key terminology associated with episomes includes "autonomous replication," which describes the self-sustaining replication process outside the using the host's replication machinery, and the "integrated state," wherein the episome becomes part of the host and is propagated during host . Plasmids represent a broader category of such extrachromosomal elements, encompassing both integrating and non-integrating types.

Key Properties

Episomes are typically composed of circular, double-stranded DNA molecules that exist extrachromosomally. These structures range in size from approximately 10 to 100 kilobases (kb), allowing them to carry substantial genetic information without overly burdening the host cell; for instance, the paradigmatic F plasmid measures about 100 kb. As dual-replicating genetic elements capable of both autonomous maintenance and chromosomal integration, episomes maintain their physical integrity through supercoiled configurations that facilitate efficient packaging and replication. When functioning autonomously, episomes exhibit low to medium copy numbers, typically 1 to 10 copies per cell, which are tightly regulated to synchronize with the host's chromosomal replication cycle. This control prevents over-replication and resource depletion, achieved through mechanisms such as via antisense or iteron-binding proteins that limit initiation at origins of replication. Such ensures stable persistence without disrupting host . Episomal stability during is bolstered by partition systems, including genes such as sopA and sopB, which actively segregate copies to daughter cells with high fidelity in low-copy episomes such as the , akin to chromosomal partitioning. These systems minimize loss rates, promoting long-term maintenance even under non-selective conditions. Episomes are generally non-essential for host viability, enabling their loss without lethal consequences, yet they often confer advantageous traits that enhance bacterial fitness in specific environments. Notably, many episomes, including resistance (R) plasmids, encode resistance genes that allow hosts to survive exposure, thereby facilitating and adaptation. This compatibility underscores their role as modular genetic accessories rather than core cellular components.

Historical Development

Discovery

The concept of the episome emerged in the mid-20th century through studies on bacterial genetics, particularly the process of conjugation in . François Jacob and Élie L. Wollman, working at the , investigated the mechanisms underlying genetic transfer during conjugation, building on earlier discoveries of sex factors in . Their research focused on the fertility factor (F factor), a genetic element that enables conjugation and was initially identified in the early 1950s as responsible for high-frequency genetic exchange between bacterial cells. In 1956, and Wollman described high-frequency recombination (Hfr) strains of E. coli, which exhibited markedly elevated rates of chromosomal transfer compared to standard F⁺ strains. Through experiments involving interrupted matings—where conjugation was mechanically disrupted using a kitchen blender to halt DNA transfer at specific times—they demonstrated that Hfr strains initiate the transfer of chromosomal DNA in a linear, time-dependent manner from a fixed origin. This observation revealed that the F factor could integrate into the bacterial at various sites, effectively linking the autonomous F plasmid-like element to the host and enabling the mobilization of adjacent chromosomal segments during conjugation. The key evidence distinguishing integrated from autonomous states came from comparing gene transfer patterns: in autonomous F⁺ cells, only the F factor itself is typically transferred rapidly, resulting in F⁺ recipients, whereas in Hfr cells, chromosomal genes are transferred sequentially following F integration, with transfer efficiency decreasing with distance from the integration point. This dual behavior prompted and Wollman to coin the term "episome" in , defining it as a genetic element capable of autonomous replication or chromosomal integration. Their findings were published in the Comptes Rendus de l'Académie des Sciences and represented a pivotal advance in bacterial , occurring shortly after the 1953 elucidation of DNA's double-helix structure by Watson and Crick, which provided the molecular framework for understanding such replicative elements.

Conceptual Evolution

In the 1960s and 1970s, the concept of episomes was refined to position them as a subset of plasmids characterized by their integrative capacity into the host , distinguishing them from purely extrachromosomal elements. This stemmed from experimental studies on R-factors, which demonstrated resistance transfer via both autonomous and integrated forms, and on bacteriophage lambda, which served as a model for reversible integration and excision mechanisms. These investigations highlighted episomes' dual replicative states, building on the initial analogy between bacterial sex factors and phage behavior established by François Jacob and Élie Wollman. Joshua Lederberg's 1952 proposal of the term "" as a generic descriptor for extrachromosomal genetic elements profoundly influenced episome classification, providing a unifying framework that encompassed integrative variants like the F factor. By the 1970s, seminal reviews further delineated conjugative episomes, which encode machinery for direct cell-to-cell transfer, from non-conjugative ones reliant on helper elements, emphasizing their differential impacts on bacterial genetics. This period marked a conceptual shift from the episome's original narrow definition—coined in 1958 by and Wollman—to a more structured subcategory within biology, driven by advances in genetic mapping and intergeneric transfer experiments. During the 1980s, the term "episome" began to extend beyond strictly bacterial contexts, occasionally describing non-integrative or eukaryotic extrachromosomal elements in vector design and viral persistence studies, though core genetic literature upheld its bacterial-specific meaning tied to integration. This broadening reflected growing applications in , yet it also prompted critiques of terminological looseness, accelerating the preference for "" as the dominant term. By , the modern consensus views "episome" as a largely historical term, frequently synonymous with integrative plasmids in prokaryotic systems, but retained in literature to denote elements with site-specific chromosomal mobility distinct from standard s. This perspective underscores episomes' foundational role in early plasmid research while acknowledging their subsumption under broader plasmid classifications based on incompatibility and function.

Molecular Structure and Replication

Genetic Composition

Episomes possess a core set of genetic elements that enable their autonomous maintenance and potential chromosomal integration. The (ori) serves as the essential cis-acting sequence directing plasmid-like replication, typically comprising AT-rich regions, iterons or direct repeats for initiator protein binding, and sometimes boxes to coordinate with host machinery. Partition loci (par), consisting of centromere-like parS sites and trans-acting proteins such as ParA and ParB, ensure equitable segregation to daughter cells during division, particularly crucial for low-copy episomes to maintain stability. Integration sites, often denoted as attachment (att) regions or homologous sequences, facilitate reversible incorporation into the host via or mechanisms. Accessory genes enhance episome functionality and adaptability within the host. Selectable markers, such as antibiotic resistance genes, allow for identification and maintenance under selective pressure, while conjugation machinery—including transfer (tra) operons with origin of transfer (oriT) and (mob) genes—supports horizontal dissemination between cells. These elements are non-essential for basic replication but confer selective advantages, such as resistance or traits. The genetic architecture of episomes exhibits a modular , with a conserved backbone encompassing the ori, par loci, and basic control elements, flanked by variable regions accommodating integration sites and accessory modules. This design permits flexibility in cargo without disrupting core functions. Core minimal episomes, comprising just the essential replication and partitioning components, though inclusion of integration and basic accessory elements extends their size; larger constructs readily accommodate inserted s, reaching tens or hundreds of kb. In viral episomes, such as temperate bacteriophages, the genetic composition includes phage-specific elements like the att site for integration and origins tailored to proteins, differing from bacterial modules.

Autonomous Replication

Autonomous replication of episomes occurs independently of chromosomal integration. In bacterial plasmid-type episomes, takes place at a dedicated (ori), where episome-encoded initiator proteins such as Rep bind to repetitive DNA sequences called iterons. This binding recruits host-encoded DNA polymerases, primarily , to unwind the DNA and synthesize new strands, with the process synchronized to the host for stable copy number maintenance. Although some episomes may encode accessory replication proteins, the core elongation relies on host machinery to ensure fidelity and efficiency. Viral episomes, such as , employ distinct mechanisms: replication initiates at oriλ, where phage proteins O and P bind and recruit host and polymerase to form a replication complex. Copy number is tightly controlled via mechanisms, in which Rep proteins, after initiating replication, dimerize and bind additional iterons to form inhibitory complexes that block re-initiation at the ori, thereby preventing over-replication and maintaining low to medium copy numbers typical of many episomes. This regulation is mediated by interactions between the ori and nearby incompatibility regions, where Rep-bound iterons create a physical or steric hindrance to further replication rounds. Stable inheritance during host cell division is achieved through active partitioning systems encoded by par genes, which include parA (an ATPase that generates dynamic gradients on the nucleoid), parB (a DNA-binding protein that clusters at the centromere-like parS site on the episome), and the parS sequence itself. These components form a partition complex that actively segregates episome copies to opposite poles of the cell during binary fission, ensuring approximately one copy per daughter cell and minimizing loss over generations. While autonomous, episomal replication depends on host cellular resources for energy and substrates, utilizing ATP to power helicases and accessory proteins during unwinding and incorporating host-supplied deoxyribonucleoside triphosphates (dNTPs) as building blocks for , distinct from the chromosomal replication fork. In eukaryotic viral episomes, such as Epstein-Barr virus, replication relies on the viral oriP and trans-acting factor EBNA-1 to tether to host chromosomes and utilize host replication machinery for persistence.

Integration Mechanisms

Chromosomal Integration

Chromosomal integration of episomes occurs primarily through homologous recombination, a process that allows the circular episomal DNA to insert into the host bacterial chromosome via a single crossover event between homologous sequences on both molecules. This mechanism results in the linearization of the episome and its covalent attachment to the chromosome, forming a stable composite structure often referred to as an "open-eight" configuration due to the duplicated homologous regions flanking the integrated element. The frequency of this integration is generally low in wild-type , typically on the order of 10^{-3} to 10^{-5} per cell, and is strongly dependent on the RecA-mediated and recombination system, which facilitates strand invasion and exchange between the homologous regions. In mutants, integration efficiency drops dramatically, often by at least a factor of 10, rendering the process rare or undetectable without external induction such as DNA damage that activates the response. Upon integration, the episomal genes become part of the host and replicate in synchrony with it, transitioning from potentially multiple autonomous copies to a single copy per cell, thereby ensuring stable without the need for independent partitioning mechanisms. This integrated state contrasts with the episome's autonomous replication, where it maintains extrachromosomal copies independently of the . Integration can have significant consequences for the host, including disruption of chromosomal genes at site due to insertion or the creation of hybrid alleles in the duplicated regions, potentially leading to loss-of-function mutations. Conversely, it enables the stable, high-level expression of episomal traits, such as antibiotic resistance or factors, as they are now under chromosomal replication control, avoiding dilution from multiple copies in the autonomous state.

Site-Specific Recombination

Site-specific recombination enables the targeted integration of episomes into the host chromosome through precise DNA strand exchanges at defined attachment sites. This process is mediated by integrase enzymes, which belong to the tyrosine recombinase family and catalyze conservative recombination events without net gain or loss of DNA. In the case of episomes derived from temperate bacteriophages, such as lambda, the integrase (Int) facilitates crossover between the phage attachment site attP on the episome and the bacterial attachment site attB on the host genome, resulting in hybrid sites attL and attR. The recombination occurs at a specific 7-base pair overlap region within these sites, where the integrase cleaves the DNA strands, forms covalent 3'-phosphotyrosine intermediates, and religates them to complete the exchange. The directionality of is tightly controlled, allowing reversible switching between integrated and extrachromosomal states. Integration is promoted by Int in complex with integration host factor (IHF), which bends the DNA to assemble the synaptic complex, while excision requires the additional action of excisionase (Xis), along with IHF and sometimes factor for inversion stimulation (Fis). Xis not only activates excisive recombination but also inhibits integration by altering the of the attP site, ensuring the process responds to physiological cues. This reversibility is a hallmark of temperate phage episomes like , where the state (integrated) can shift to the (excised). Regulation occurs through environmental signals, such as varying levels of IHF and Fis proteins in the host, as well as immunity repressors like the lambda CI protein, which modulates Int expression to maintain lysogenic stability. Compared to , exhibits markedly higher efficiency, achieving up to 100% integration in lysogeny under optimal conditions and . This precision stems from the enzyme's specificity for short, defined sequences—attB is only 21 base pairs—allowing rapid and error-free events without extensive homology searching. At the molecular level, the process proceeds through a intermediate formed after the first strand exchange (top strands), which is then resolved by a second cleavage and religation (bottom strands) catalyzed by the integrase tetramer. Critically, this resolution is independent of the host protein, relying instead on the recombinase's catalytic domain and accessory factors to enforce branch migration and strand selectivity.

Episomes in Prokaryotes

Bacterial Examples

One prominent bacterial episome is the F plasmid (also known as the fertility factor) in Escherichia coli, a conjugative plasmid approximately 100 kb in size that encodes genes for sex pilus formation via the tra operon, facilitating bacterial conjugation. This episome can integrate into the E. coli chromosome through recombination at insertion sequence (IS) elements, forming high-frequency recombination (Hfr) strains that enable partial chromosome transfer during conjugation. Resistance (R) plasmids represent a class of conjugative plasmids prevalent in , particularly those conferring resistance through clustered genes on an r-determinant segment, often paired with a resistance transfer factor (RTF) that provides conjugation and replication functions similar to the . These plasmids, transferable via conjugation, can in some cases integrate into the host , allowing stable inheritance of resistance traits in Gram-negative species. The P1 of E. coli serves as a temperate , with a of approximately 94 kb that typically persists as a low-copy-number in lysogenic cells, maintained at about one copy per through a partitioning system. Under certain conditions, it can integrate into the bacterial via at attachment (att) sites, though it predominantly replicates extrachromosomally. Integration mechanisms, such as those involving IS elements or att sites, underlie the dual lifestyles of these episomes in enabling both autonomous replication and chromosomal association. Such episomes are commonly found in , especially within the family, where conjugative elements like F and R plasmids contribute to genetic diversity and adaptability.

Role in Horizontal Gene Transfer

Episomes play a central role in among prokaryotes, primarily through the process of , which enables the direct transfer of genetic material between donor and recipient cells. In this mechanism, an episome like the F plasmid in encodes genes for forming a conjugative , a proteinaceous bridge that connects the donor and recipient bacteria, facilitating DNA passage. Once contact is established, the episome initiates rolling-circle replication within the donor cell, nicking its DNA at the origin of transfer and displacing a single-stranded copy that is threaded through the pilus into the recipient. This single-stranded transfer ensures efficient dissemination of the episomal DNA, which is then replicated into double-stranded form in the recipient, converting it into a donor capable of further propagation. When an episome integrates into the bacterial chromosome, forming a high-frequency recombination (Hfr) strain, it mobilizes adjacent chromosomal DNA for transfer during conjugation. Integration occurs via homologous recombination between the episome and the chromosome, positioning the origin of transfer such that linear segments of chromosomal DNA are transferred sequentially starting from the integration site. The process continues via rolling-circle replication until the conjugative bridge typically breaks prematurely, resulting in partial chromosomal transfer rather than complete genome exchange. This mobilization allows for the spread of host chromosomal genes alongside episomal elements, broadening the scope of genetic exchange beyond self-replicating plasmids. Episome-mediated conjugation accelerates the dissemination of adaptive traits, such as antibiotic resistance and virulence factors, across bacterial populations by enabling rapid gene sharing even among distantly related . For instance, conjugative episomes carrying multiple resistance genes can transfer en masse, converting susceptible into resistant ones in high-density environments like biofilms or the . This horizontal spread has been documented in clinical isolates, where episomes contribute to outbreaks of multidrug-resistant pathogens by propagating resistance cassettes at rates far exceeding vertical inheritance. From an evolutionary perspective, episomes enhance bacterial adaptability in dynamic environments, such as those altered by use, by facilitating the acquisition of beneficial alleles without relying on alone. In the post- era, this transfer mechanism has driven the emergence of resistant lineages, allowing populations to evade selective pressures and colonize new niches. By promoting and recombination, episomes thus act as key drivers of prokaryotic , underscoring their significance in microbial and challenges.

Episomes in Eukaryotes

Viral Episomes

Viral episomes represent a key mechanism by which certain eukaryotic viruses, particularly herpesviruses, establish persistent latent infections without integrating into the host genome. In this state, the viral genome exists as an extrachromosomal, circular DNA molecule in the host cell nucleus, replicating autonomously in synchrony with the host cell cycle using host machinery. This form allows the virus to evade immune detection while maintaining long-term presence in the host. The Epstein-Barr virus (EBV), a gammaherpesvirus, exemplifies viral episomes during latency, where its linear genome circularizes upon entry into the nucleus of infected B lymphocytes, forming a stable extrachromosomal episome. This episome replicates once per via the host , directed by the viral origin of replication (oriP) and the essential EBNA-1 protein, ensuring persistence without lytic replication. EBV latency as an episome is central to its association with and certain cancers. Similarly, (KSHV), also known as human herpesvirus 8, maintains its genome as circular episomes in latently infected B cells and endothelial cells, utilizing terminal repeat sequences analogous to EBV's oriP for replication and retention. The viral latency-associated nuclear antigen (LANA) facilitates episome persistence by binding these repeats and tethering the genome to host mitotic spindles, promoting segregation during . KSHV episomal latency contributes to oncogenesis, driving diseases such as and primary effusion through sustained expression of latent genes. A critical aspect of episome maintenance in these viruses involves protein-mediated tethering to host chromosomes, as seen with EBV's EBNA-1, which binds oriP and interacts with host chromatin to ensure equitable distribution to daughter cells during . This mechanism supports stable propagation in dividing cells. Unlike prokaryotic episomes, viral episomes in eukaryotes exhibit enhanced stability in non-dividing or quiescent cells, such as long-lived memory B cells, and do not rely on conjugation for transfer, instead depending on cell-to-cell spread or reactivation.

Engineered Episomes

Engineered episomes draw inspiration from natural viral episomes but incorporate synthetic elements to enable stable, non-integrating expression in eukaryotic cells, particularly for research and therapeutic applications in mammalian systems. These vectors prioritize nuclear retention, autonomous replication, and resistance to dilution during , avoiding the risks of genomic integration such as . Design principles focus on incorporating specific DNA sequences that mimic chromosomal attachment and replication origins, allowing persistence over multiple cell generations without selection. Scaffold/matrix attachment region (S/MAR)-based episomes utilize S/MAR sequences, such as the one derived from the human β-interferon gene, to anchor the vector to the nuclear matrix, facilitating nuclear retention and replication in mammalian cells. These elements promote mitotic stability by associating with the chromosomal , enabling episomal maintenance for up to 100 cell generations in cell lines like without integration. Enhancements, such as combining S/MAR with the β-globin replicator, have improved efficiency in hematopoietic progenitor cells to approximately 32% and increased plasmid copy numbers by 50%, supporting long-term expression in applications like gene correction. Epstein-Barr virus (EBV)-derived vectors rely on the and EBNA-1 protein to achieve stable episomal maintenance, particularly in studies within human cell lines. The oriP sequence directs replication during the , while EBNA-1 binds to it to tether the episome to host chromosomes, ensuring segregation during and persistence for over a month without integration. These vectors have been optimized into single-component systems, such as pCEP4 derivatives, that maintain low copy numbers (5-15 per cell) and support consistent expression in proliferating cells like Jurkat T cells. A primary challenge for engineered episomes is epigenetic silencing over time, driven by heterochromatin formation and high CpG content, which leads to loss of transgene expression after initial transduction. In the 2020s, advances have addressed this through incorporation of insulator elements and CpG-depleted designs; for instance, CpG-reduced episomal vectors like pEPito, which has 60% fewer CpG motifs than earlier designs, reduce silencing by minimizing bacterial DNA motifs, enhancing longevity and expression levels in primary cells. Nano-formulated S/MAR vectors further improve persistence, doubling colony formation efficiency in HEK293 cells while exhibiting low toxicity. In chimeric antigen receptor (CAR)-T cell engineering, S/MAR-based episomes enable transient yet prolonged transgene expression without integration risks, providing a safer alternative to viral integrating vectors. Non-integrating lentiviral vectors with S/MAR maintain CAR expression and cytotoxic function comparable to standard lentivirals, with stable GFP and CD19-CAR persistence over weeks in transduced T cells, effectively targeting CD19+ tumors in vivo while showing no detectable genotoxicity.

Biological Significance and Applications

In Microbial Evolution

Episomes serve as that expand the microbial by enabling the transfer and integration of accessory genes across populations, thereby accelerating evolutionary adaptation through recombination events. In natural marine environments, such as those inhabited by , episomes including plasmids and temperate phages exhibit promiscuous recombination patterns, connecting diverse gene families and facilitating the rapid assembly of functional modules like type IV secretion systems and maintenance genes. This mobility enhances genetic diversity beyond chromosomal alone, allowing to acquire traits for environmental responsiveness without relying solely on vertical . In and , episomes promote niche adaptation by carrying genes that confer competitive advantages or virulence factors. For instance, nontransmissible plasmids in species often encode genes, supporting iron acquisition in nutrient-limited symbiotic interactions with hosts. Similarly, in , the CTX filamentous phage functions as an episome in its lysogenic state, harboring the genes (ctxAB) that enable pathogenic colonization of the human gut, thus facilitating transmission and survival in diverse ecological niches. These examples illustrate how episomes drive the evolution of symbiotic relationships and disease-causing capabilities in microbial communities. Episomes influence microbial under selective pressures, notably the widespread use of since the , which has driven the proliferation of resistance-encoding elements. The discovery and mass production of penicillin in the mid- initiated strong selective forces favoring harboring R-plasmids—conjugative episomes carrying resistance genes—leading to their rapid dissemination across bacterial lineages. This post- shift resulted in uneven episome distribution, with resistance plasmids becoming prevalent in clinical and environmental populations, altering community structures and enhancing survival in antibiotic-exposed habitats. via these episomes has been a key mechanism in this adaptive response. Metagenomic analyses highlight the long-term evolutionary impact of episomes, demonstrating their substantial contributions to bacterial s through the of accessory genes. In gut microbiomes, for example, reconstructed plasmids from metagenomic data reveal diverse systems that enrich the 's flexible gene repertoire, often encoding metabolic or resistance functions that shape species-wide adaptability. Studies of populations further show high episome turnover, with multimember families and singletons indicating ongoing gene flux that sustains openness over evolutionary timescales. This episomal input underscores their role in maintaining microbial diversity and resilience in dynamic ecosystems.

In Gene Therapy and Biotechnology

Non-integrating episomal vectors have emerged as a safer alternative in by minimizing the risk of associated with integrating vectors, such as retroviruses, which can disrupt host genes and lead to oncogenesis. These vectors maintain the therapeutic extrachromosomally, allowing transient or prolonged expression without permanent genomic alteration. In hemophilia treatment, (AAV)-based episomal vectors have been pivotal in clinical trials during the 2020s, delivering or IX genes to hepatocytes for sustained clotting factor production; for instance, trials like those for etranacogene dezaparvovec demonstrated durable expression with reduced bleeding events over multiple years, avoiding integration-related complications observed in earlier lentiviral approaches. This design has improved safety profiles, with low and no reported in long-term follow-ups. In industrial , episomal expression systems enable high-yield recombinant in mammalian cell lines without stable genomic integration, preserving cell line integrity for repeated use and reducing regulatory hurdles. The Epi-CHO system, utilizing autonomously replicating plasmids in ovary () cells, supports transient yet high-level expression of therapeutic proteins like monoclonal antibodies, achieving titers of 75 mg/L in optimized cultures while avoiding selection pressure and genetic instability. Similarly, in yeast hosts such as pastoris, episomal vectors based on 2μ origins facilitate rapid screening and scale-up for biologics production, offering advantages over integrative methods by allowing easy vector swapping and minimizing off-target effects. These approaches have streamlined for and enzymes, with episomal systems contributing to biopharmaceutical yields in transient platforms. Episomal plasmids are widely employed for transient delivery of CRISPR-Cas9 components in bacterial engineering, enabling precise without persistent nuclease activity that could induce off-target mutations. By expressing and guide RNAs from non-integrating plasmids, such as those with low-copy replicons in or species, researchers achieve high-efficiency knockouts or insertions for optimization, with expression limited to hours or days before plasmid dilution during . This strategy has been instrumental in engineering for production and resistance studies, where episomal systems like pCas9 plasmids yield high editing rates in targeted loci. As of , episome-based technologies continue to advance in vaccine development, with DNA vectors entering late-stage clinical trials for infectious diseases and cancers, though full FDA approvals remain pending for human use beyond veterinary applications; notable progress includes hybrid vectors combining /matrix attachment regions (S/MAR) with viral elements to enhance long-term episomal stability and expression persistence in dividing cells. These hybrids address key challenges like episome loss during by promoting nuclear retention and epigenetic silencing resistance, achieving stable levels for over 100 population doublings in preclinical models. Engineered episomes thus represent versatile tools for next-generation therapies.

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