Lentiviral vector determinants of anion-exchange chromatography elution heterogeneity.

The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G]) did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).

Safety of non-replicative and oncolytic replication-selective HSV vectors.

Herpes simplex virus type 1 (HSV-1) is a DNA virus and human pathogen used to construct promising therapeutic vectors. HSV-1 vectors fall into two classes: replication-selective oncolytic vectors for cancer therapy and defective non-replicative vectors for gene therapy. Vectors from each class can accommodate ≥30 kb of inserts, have been approved clinically, and demonstrate a relatively benign safety profile. Despite oncolytic HSV (oHSV) replication in tumors and elicited immune responses, the virus is well tolerated in cancer patients. Current non-replicative vectors elicit only limited immune responses. Seropositivity and immune responses against HSV-1 do not eliminate either the vector or infected cells, and the vectors can therefore be re-administered. In this review we highlight vectors that have been translated to the clinic and host-virus immune interactions that impact on the safety and efficacy of HSVs.

Phage Display as a Medium for Target Therapy Based Drug Discovery, Review and Update.

Phage libraries are now amongst the most prominent approaches for the identification of high-affinity antibodies/peptides from billions of displayed phages in a specific library through the biopanning process. Due to its ability to discover potential therapeutic candidates that bind specifically to targets, phage display has gained considerable attention in targeted therapy. Using this approach, peptides with high-affinity and specificity can be identified for potential therapeutic or diagnostic use. Furthermore, phage libraries can be used to rapidly screen and identify novel antibodies to develop immunotherapeutics. The Food and Drug Administration (FDA) has approved several phage display-derived peptides and antibodies for the treatment of different diseases. In the current review, we provided a comprehensive insight into the role of phage display-derived peptides and antibodies in the treatment of different diseases including cancers, infectious diseases and neurological disorders. We also explored the applications of phage display in targeted drug delivery, gene therapy, and CAR T-cell.

Prenatal AAV9-GFP administration in fetal lambs results in transduction of female germ cells and maternal exposure to virus.

Prenatal somatic cell gene therapy (PSCGT) could potentially treat severe, early-onset genetic disorders such as spinal muscular atrophy (SMA) or muscular dystrophy. Given the approval of adeno-associated virus serotype 9 (AAV9) vectors in infants with SMA by the U.S. Food and Drug Administration, we tested the safety and biodistribution of AAV9-GFP (clinical-grade and dose) in fetal lambs to understand safety and efficacy after umbilical vein or intracranial injection on embryonic day 75 (E75) . Umbilical vein injection led to widespread biodistribution of vector genomes in all examined lamb tissues and in maternal uteruses at harvest (E96 or E140; term = E150). There was robust GFP expression in brain, spinal cord, dorsal root ganglia (DRGs), without DRG toxicity and excellent transduction of diaphragm and quadriceps muscles. However, we found evidence of systemic toxicity (fetal growth restriction) and maternal exposure to the viral vector (transient elevation of total bilirubin and a trend toward elevation in anti-AAV9 antibodies). There were no antibodies against GFP in ewes or lambs. Analysis of fetal gonads demonstrated GFP expression in female (but not male) germ cells, with low levels of integration-specific reads, without integration in select proto-oncogenes. These results suggest potential therapeutic benefit of AAV9 PSCGT for neuromuscular disorders, but warrant caution for exposure of female germ cells.

Dual-mRNA Delivery Using Tumor Cell Lysate-Based Multifunctional Nanoparticles as an Efficient Colon Cancer Immunogene Therapy.

Background: Messenger RNA (mRNA)-based immunogene therapy holds significant promise as an emerging tumor therapy approach. However, the delivery efficiency of existing mRNA methods and their effectiveness in stimulating anti-tumor immune responses require further enhancement. Tumor cell lysates containing tumor-specific antigens and biomarkers can trigger a stronger immune response to tumors. In addition, strategies involving multiple gene therapies offer potential optimization paths for tumor gene treatments. Methods: Based on the previously developed ideal mRNA delivery system called DOTAP-mPEG-PCL (DMP), which was formed through the self-assembly of 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) and methoxypoly (ethylene glycol)-b-poly (ε-caprolactone) (mPEG-PCL), we introduced a fused cell-penetrating peptide (fCPP) into the framework and encapsulated tumor cell lysates to form a novel nanovector, termed CLSV system (CLS: CT26 tumor cell lysate, V: nanovector). This system served a dual purpose of facilitating the delivery of two mRNAs and enhancing tumor immunogene therapy through tumor cell lysates. Results: The synthesized CLSV system had an average size of 241.17 nm and a potential of 39.53 mV. The CLSV system could not only encapsulate tumor cell lysates, but also deliver two mRNAs to tumor cells simultaneously, with a transfection efficiency of up to 60%. The CLSV system effectively activated the immune system such as dendritic cells to mature and activate, leading to an anti-tumor immune response. By loading Bim-encoded mRNA and IL-23A-encoded mRNA, CLSV/Bim and CLSV/IL-23A complexes were formed, respectively, to further induce apoptosis and anti-tumor immunity. The prepared CLSV/dual-mRNA complex showed significant anti-cancer effects in multiple CT26 mouse models. Conclusion: Our results suggest that the prepared CLSV system is an ideal delivery system for dual-mRNA immunogene therapy.

Genome Editing Approaches Using Zinc Finger Nucleases (ZFNs) for the Treatment of Motor Neuron Diseases.

Motor neuron disorders are diseases that can be passed through generations by heredity or they occur due to spontaneous mutations in the gene. These are the disorders that weaken the connection between motor neurons and the muscles, due to this the coordination between the neurons and muscles gets disturbed and thereby the actions become abnormal, every year millions of people around the world suffer from these different types of motor neuron disorders. Till now there is no proper known treatment for this type of disorder, there is active research work going on to treat these diseases permanently. Some gene therapy treatments are giving promising results in the treatment of these diseases, specifically, genetic modification techniques are the front liners, and many types of nucleases are doing their work to replace the mutated gene with a functional one. Zinc finger nucleases (ZFNs) are one of them with good disease treatment potential with accurate and desirable effects. In this review, we note the complete information about ZFNs and their drawbacks along with their future prospective in gene therapy and also shortly with other types of nucleases-mediated gene therapies. There also some factors that influence the gene therapy treatment are also noted along with some detailed information.

Cationic Polythiophene as Gene Carrier and Sonosensitizer for Sonodynamic Synergic Gene Therapy of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal and highly malignant tumors. Sonodynamic therapy (SDT) is a new cancer treatment method. One of its unique advantages lies in the treatment of deep tumors due to its excellent tissue penetration ability caused by ultrasound (US). However, most sonosensitizers suffer from weak sonodynamic activity and poor tumor-targeting ability. In addition, small interfering RNA (siRNA) is a promising anticancer drug, and the efficacy of siRNA-based gene therapy largely depends on the cell impermeability of the gene carrier. Here, we designed and synthesized a cationic polythiophene derivative (PT2) that can be used as a siRNA carrier for gene therapy. Moreover, PT2 could generate singlet oxygen (1O2) and hydroxyl radicals (O2•-) under US irradiation, which suggests that PT2 could be used for SDT. Our study discovered that NUDT1 promoted HCC proliferation and inhibited intracellular ROS production. Therefore, si-NUDT1 was designed and synthesized. NUDT1 silencing can inhibit the proliferation of tumor cells and increase the production of intracellular ROS to further improve the efficacy of SDT. Then, si-NUDT1 assembled with PT2 and DSPE-PEG-FA to prepare a novel tumor-targeting nanodrug (PT2-siRNA@PEG-FA) for synergic SDT and gene therapy of HCC.

Smart Physicochemical-triggered Chitosan-based Nanogels for siRNA Delivery and Gene Therapy: A Focus on Emerging Strategies and Paradigms for Cancer Therapy.

Cancer therapy has seen significant advancements in recent years, with the emergence of RNA interference (RNAi) as a promising strategy for targeted gene silencing. However, the successful delivery of small interfering RNA (siRNA) to cancer cells remains a challenge. Chitosan nanoparticles (CSNPs) can be derived from the natural polysaccharide chitin sources. CSNPs have gained considerable attention as a potential solution to encapsulate siRNA due to their biocompatibility, and biodegradability. This article explores the application of CSNPs for siRNA delivery in cancer therapy. Firstly, it discusses the significance of siRNA in gene regulation and highlights its potential to selectively silence oncogenes or tumor suppressor genes, making it a powerful tool in cancer treatment. The obstacles associated with effective siRNA delivery, such as degradation by nucleases and poor cellular uptake, are also addressed. Next, the focus shifts to the unique properties of CSNPs that make them attractive for siRNA delivery. The discussion revolves around how chitosan can interact electrostatically with siRNA to create stable complexes, as well as the controlled release of siRNA from CSNPs. This controlled release ensures sustained and efficient delivery of siRNA to cancer cells, maximizing therapeutic efficacy. Moreover, the biocompatibility and biodegradability of CSNPs make them ideal for in vivo applications. Different approaches to modifying and functionalizing surfaces are investigated by emphasizing on enhancement of stability and targeting abilities of CSNPs in cancer treatment. Registered trials for CS and siRNA are summarized, along with ongoing investigations into various applications of chitosan in medical treatments. Overall, the application of CSNPs in siRNA delivery for cancer therapy holds great promise and offers a potential solution to overcome the challenges associated with RNAi-based treatments. Continued advancements in this field will likely lead to improved targeted therapies with reduced side effects, ultimately benefitting cancer patients worldwide.

Advances and challenges in gene therapy strategies for pediatric cancer: a comprehensive update.

Pediatric cancers represent a tragic but also promising area for gene therapy. Although conventional treatments have improved survival rates, there is still a need for targeted and less toxic interventions. This article critically analyzes recent advances in gene therapy for pediatric malignancies and discusses the challenges that remain. We explore the innovative vectors and delivery systems that have emerged, such as adeno-associated viruses and non-viral platforms, which show promise in addressing the unique pathophysiology of pediatric tumors. Specifically, we examine the field of chimeric antigen receptor (CAR) T-cell therapies and their adaptation for solid tumors, which historically have been more challenging to treat than hematologic malignancies. We also discuss the genetic and epigenetic complexities inherent to pediatric cancers, such as tumor heterogeneity and the dynamic tumor microenvironment, which pose significant hurdles for gene therapy. Ethical considerations specific to pediatric populations, including consent and long-term follow-up, are also analyzed. Additionally, we scrutinize the translation of research from preclinical models that often fail to mimic pediatric cancer biology to the regulatory landscapes that can either support or hinder innovation. In summary, this article provides an up-to-date overview of gene therapy in pediatric oncology, highlighting both the rapid scientific progress and the substantial obstacles that need to be addressed. Through this lens, we propose a roadmap for future research that prioritizes the safety, efficacy, and complex ethical considerations involved in treating pediatric patients. Our ultimate goal is to move from incremental advancements to transformative therapies.

Lipid nanoparticles-based RNA therapies for breast cancer treatment.

Breast cancer (BC) prevails as a major burden on global healthcare, being the most prevalent form of cancer among women. BC is a complex and heterogeneous disease, and current therapies, such as chemotherapy and radiotherapy, frequently fall short in providing effective solutions. These treatments fail to mitigate the risk of cancer recurrence and cause severe side effects that, in turn, compromise therapeutic responses in patients. Over the last decade, several strategies have been proposed to overcome these limitations. Among them, RNA-based technologies have demonstrated their potential across various clinical applications, notably in cancer therapy. However, RNA therapies are still limited by a series of critical issues like off-target effect and poor stability in circulation. Thus, novel approaches have been investigated to improve the targeting and bioavailability of RNA-based formulations to achieve an appropriate therapeutic outcome. Lipid nanoparticles (LNPs) have been largely proven to be an advantageous carrier for nucleic acids and RNA. This perspective explores the most recent advances on RNA-based technology with an emphasis on LNPs' utilization as effective nanocarriers in BC therapy and most recent progresses in their clinical applications.

Molecular therapy and nucleic acid adeno-associated virus-based gene therapy delivering combinations of two growth-associated genes to MPS IVA mice.

Mucopolysaccharidosis type IVA (MPS IVA) is caused by a deficiency of the galactosamine (N-acetyl)-6-sulfatase (GALNS) enzyme responsible for the degradation of specific glycosaminoglycans (GAGs). The progressive accumulation of GAGs leads to various skeletal abnormalities (short stature, hypoplasia, tracheal obstruction) and several symptoms in other organs. To date, no treatment is effective for patients with bone abnormalities. To improve bone pathology, we propose a novel combination treatment with the adeno-associated virus (AAV) vectors expressing GALNS enzyme and a natriuretic peptide C (CNP; NPPC gene) as a growth-promoting agent for MPS IVA. In this study, an MPS IVA mouse model was treated with an AAV vector expressing GALNS combined with another AAV vector expressing NPPC gene, followed for 12 weeks. After the combination therapy, bone growth in mice was induced with increased enzyme activity in tissues (bone, liver, heart, lung) and plasma. Moreover, there were significant changes in bone morphology in CNP-treated mice with increased CNP activity in plasma. Delivering combinations of CNP and GALNS gene therapies enhanced bone growth in MPS IVA mice more than in GALNS gene therapy alone. Enzyme expression therapy alone fails to reach the bone growth region; our results indicate that combining it with CNP offers a potential alternative.

Phase 1/2 AAV5-hRKp.RPGR (Botaretigene Sparoparvovec) Gene Therapy: Safety and Efficacy in RPGR-associated X-linked Retinitis Pigmentosa.

PURPOSE: To assess the safety and efficacy of AAV5-hRKp.RPGR in participants with retinitis pigmentosa GTPase regulator (RPGR)-associated X-linked retinitis pigmentosa (XLRP). DESIGN: Open-label, phase 1/2 dose escalation/expansion study (NCT03252847). METHODS: Males (≥5 years old) with XLRP-RPGR were evaluated. In the dose escalation phase, subretinal AAV5-hRKp.RPGR (low: 1.0×1011 vg/ml; intermediate: 2.0×1011 vg/ml; high: 4.0×1011 vg/ml) was administered to the poorer-seeing eye (n = 10). Dose confirmation (intermediate dose) was carried out in 3 pediatric participants. In the dose expansion phase, 36 participants were randomized 1:1:1 to immediate (low or intermediate dose) or deferred (control) treatment. The primary outcome was safety. Secondary efficacy outcomes included static perimetry, microperimetry, vision-guided mobility, best corrected visual acuity, and contrast sensitivity. Safety and efficacy outcomes were assessed for 52 weeks for immediate treatment participants and 26 weeks for control participants. RESULTS: AAV5-hRKp.RPGR was safe and well tolerated, with no reported dose-limiting events. Most adverse events (AEs) were transient and related to the surgical procedure, resolving without intervention. Two serious AEs were reported with immediate treatment (retinal detachment, uveitis). A third serious AE (increased intraocular pressure) was reported outside the reporting period. All ocular inflammation-related AEs responded to corticosteroids. Treatment with AAV5-hRKp.RPGR resulted in improvements in retinal sensitivity and functional vision compared with the deferred group at Week 26; similar trends were observed at Week 52. CONCLUSIONS: AAV5-hRKp.RPGR demonstrated an anticipated and manageable AE profile through 52 weeks. Safety and efficacy findings support investigation in a phase 3 trial.

Conventional and microfluidic methods: Design and optimization of lipid-polymeric hybrid nanoparticles for gene therapy.

Gene therapy holds significant promise as a therapeutic approach for addressing a diverse range of diseases through the suppression of overexpressed proteins and the restoration of impaired cell functions. Developing a nanocarrier that can efficiently load and release genetic material into cells remains a challenge. The primary goal of this study is to develop formulations aimed to enhance the therapeutic potential of GapmeRs through technological approaches. To this end, lipid-polymeric hybrid nanoparticles (LPHNPs) with PLGA, DC-cholesterol, and DOPE-mPEG2000 were produced by conventional single-step nanoprecipitation (SSN) and microfluidic (MF) methods. The optimized nanoparticles by SSN have a size of 149.9 ± 18.07 nm, a polydispersity index (PdI) of 0.23 ± 0.02, and a zeta potential of (ZP) of 29.34 ± 2.44 mV, while by MF the size was 179.8 ± 6.3, a PdI of 0.24 ± 0.01, and a ZP of 32.25 ± 1.36 mV. Furthermore, LPHNPs prepared with GapmeR-protamine by both methods exhibit a high encapsulation efficiency of approximately 90%. The encapsulated GapmeR is completely released in 24 h. The LPHNP suspensions are stable for up to 6 h in 10% FBS at pH 5.4 and 7.4. By contrast, LPHNPs remain stable in suspension in 4.5% albumin at pH 7.4 for 24 h. Additionally, LPHNPs were successfully freeze-dried using trehalose in the range of 2.5-5% as cryoprotectant The LPHNPs produced by MF and SSN increase, 6 and 12 fold respectively, GapmeR cell uptake, and both of them reduce by 60-70% expression of Tob1 in 48 h.Our study demonstrates the efficacy of the developed LPHNPs as carriers for oligonucleotide delivery, offering valuable insights for their scale up production from a conventional bulk methodology to a high-throughput microfluidic technology.

An evaluation of nadofaragene firadenovec-vncg for the treatment of high-risk BCG-unresponsive non-muscle-invasive bladder cancer.

INTRODUCTION: BCG-unresponsive non-muscle invasive bladder cancer (NMIBC) represent a significant therapeutic challenge in the treatment of bladder cancer. Nadofaragene firadenovec, represents a breakthrough in this area, offering a novel approach for the treatment of BCG-unresponsive NMIBC. AREAS COVERED: This overview explores the historical development of nadofaragene firadenovec, assessing its efficacy and safety, and discusses future NMIBC therapy directions. EXPERT OPINION: Patients with high grade NMIBC who are BCG unresponsive will have a growing number of treatment alternatives to bladder removal. Nadofaragene firadenovec offers good short-term efficacy but lacks significant durability for most patients. Its strengths include ease of administration and low risk of adverse events. This will need to balance with risk of progression and cost. Furthermore, the likely approval of other agents will require consideration of which therapy to use and for which patient. The need for biomarkers to tailor treatment choices to individual patient needs is becoming more critical. The treatment field is rapidly advancing, with several Phase 3 single-arm trials underway, indicating a potential broader range of treatment options for NMIBC. Further research will be necessary to determine the optimal choice for patients.

AAV9-Mediated Intra-Striatal Delivery of GNAO1 Reduces Hyperlocomotion in Gnao1 Heterozygous R209H Mutant Mice.

Mutations in the GNAO1 gene, which encodes the abundant brain G-protein Gαo, result in neurologic disorders characterized by developmental delay, epilepsy, and movement abnormalities. There are over 50 mutant alleles associated with GNAO1 disorders; the R209H mutation results in dystonia, choreoathetosis, and developmental delay without seizures. Mice heterozygous for the human mutant allele (Gnao1 +/R209H) exhibit hyperactivity in open field tests but no seizures. We developed self-complimentary adeno-associated virus vectors (scAAV9) expressing two splice variants of human GNAO1 Gαo isoforms 1 (GoA, GNAO1.1) and 2 (GoB, GNAO1.2). Bilateral intra-striatal injections of either scAAV9-GNAO1.1 or scAAV9-GNAO1.2 significantly reversed mutation-associated hyperactivity in open field tests. GNAO1 overexpression did not increase seizure susceptibility, a potential side-effect of GNAO1 vector treatment. This represents the first report of successful preclinical gene therapy for GNAO1 encephalopathy applied in vivo Further studies are needed to uncover the molecular mechanism that results in behavior improvements after scAAV9-mediated Gαo expression and to refine the vector design. Significance Statement GNAO1 mutations cause a spectrum of developmental, epilepsy, and movement disorders. Here, we show that intra-striatal delivery of scAAV9-GNAO1 to express the wild-type Gαo protein reduces the hyperactivity of the Gnao1 +/R209H mouse model, which carries one of the most common movement disorder-associated mutations. This is the first report of a gene therapy for GNAO1 encephalopathy applied in vivo on a patient-allele model.

Enkephalin Rescues Temporomandibular Joint Pain-Related Behavior in Rats.

Temporomandibular joint disorders include a variety of clinical syndromes that are difficult to manage if associated with debilitating severe jaw pain. Thus, seeking additional experimental therapies for temporomandibular joint pain reduction is warranted. Targeted enkephalin gene therapy approaches provide clear promise for pain control. The studies detailed here indicate significant analgesia and protection of joint tissue are provided after injection of an overexpression viral vector gene therapy near the joint. The viral vector gene therapy described provides overexpression of naturally occurring opioid peptides after its uptake by trigeminal nerve endings. The viral vectors act as independent "minipump" sources for the opioid peptide synthesis in the neuronal cytoplasm producing the intended biological function, reduction of pain, and tissue repair. The antinociceptive effects provided with this delivery method of opioid expression persist for over 4 weeks. This is coincident with the expected time frame for the duration of the transgene overproduction of the endogenous opioid peptide before its diminution due to dormancy of the virus. These experimental studies establish a basis for the use of replication-defective herpes simplex type 1-based gene therapy for severe chronic inflammatory temporomandibular joint destruction and pain. As innovative means of significantly reducing joint inflammation and preserving tissue architecture, gene therapies may extend their clinical usefulness for patients with temporomandibular joint disorders.

Advances and opportunities in process analytical technologies for viral vector manufacturing.

Viral vectors are an emerging, exciting class of biologics whose application in vaccines, oncology, and gene therapy has grown exponentially in recent years. Following first regulatory approval, this class of therapeutics has been vigorously pursued to treat monogenic disorders including orphan diseases, entering hundreds of new products into pipelines. Viral vector manufacturing supporting clinical efforts has spurred the introduction of a broad swath of analytical techniques dedicated to assessing the diverse and evolving panel of Critical Quality Attributes (CQAs) of these products. Herein, we provide an overview of the current state of analytics enabling measurement of CQAs such as capsid and vector identities, product titer, transduction efficiency, impurity clearance etc. We highlight orthogonal methods and discuss the advantages and limitations of these techniques while evaluating their adaptation as process analytical technologies. Finally, we identify gaps and propose opportunities in enabling existing technologies for real-time monitoring from hardware, software, and data analysis viewpoints for technology development within viral vector biomanufacturing.

Transdermal gene delivery.

Gene delivery has revolutionized conventional medical approaches to vaccination, cancer, and autoimmune diseases. However, current gene delivery methods are limited to either intravenous administration or direct local injections, failing to achieve well biosafety, tissue targeting, drug retention, and transfection efficiency for desired therapeutic outcomes. Transdermal drug delivery based on various delivery strategies can offer improved therapeutic potential and superior patient experiences. Recently, there has been increased foundational and clinical research focusing on the role of the transdermal route in gene delivery and exploring its impact on the efficiency of gene delivery. This review introduces the recent advances in transdermal gene delivery approaches facilitated by drug formulations and medical devices, as well as discusses their prospects.

Plant-derived Exosomes: Pioneering Breakthroughs in Therapeutics, Targeted Drug Delivery, and Regenerative Medicine.

Small extracellular vesicles called exosomes, which cells release, have drawn a lot of attention recently because of their ability to serve as therapeutic delivery systems for drugs and regenerative medicine applications. The investigation of plant-based exosomes as a cutting-edge platform for drug administration has emerged as an enticing research topic. A summary of the pharmaceutical feasibility of exosomes generated from plants and their uses in drug delivery along with regenerative medicine are the goals of this review study. Plant exosomes can be combined into nanoparticlebased medication delivery systems to increase their stability, targeting, and cargo delivery capabilities. By loading plant exosomes with therapeutic compounds and encapsulating them within nanoparticles, controlled release and targeted distribution to specific cells or tissues may be achieved. In gene therapy, plant exosomes can be modified to carry nucleic acids like plasmid DNA, siRNA, or miRNA. Effective gene delivery and therapeutic gene expression regulation can be accomplished by encasing nucleic acids in exosomes or surface-modifying exosomes to improve their interaction with target cells. In this review, we through the history and features of plant exosomes, examine how they differ from mammalian exosomes, and consider how they may be used for gene therapy, tissue regeneration, and targeted medication delivery. The difficulties and prospects for creating exosomebased plant medicines are also explored.