Gene transfer is a process of transferring or incorporating genetic material from one organism to another or between cells of the same organism¹.

Gene transfer can encode traits or characteristics in the recipient, leading to changes in the genetic makeup of the recipient organism. This plays a role in evolution, for example, the development of antibiotic resistance in bacteria by the transfer of antibiotic resistance genes^2,3.

Gene transfer is an essential mechanism needed for both natural processes and human-engineered applications. It contributes to genetic diversity, reproduction, and adaptation naturally in organisms like bacteria and archaea. It also plays an important role in biotechnology, such as the DNA SARS-CoV-2vaccine, containing a plasmid vector that encodes the spike (S) protein of the same virus, including its receptor binding domain (RBD), which attaches to the human ACE-2 receptor⁴. It is also used for creating genetically modified organisms (GMOs) for enhanced function in agriculture, for example, Bt crops and pharmaceuticals such as exagamglogene autotemcel have been approved for treating sickle cell disease in patients aged 12 and older⁵.

In medicine, gene transfer is useful in gene therapy and vaccine development, such as the DNA SARS-CoV-2 vaccine^6,7. Further, it may be used to aid in environmental solutions like bioremediation, solving challenges in health and sustainability, such as cleaning up oil spills⁸.

Types of gene transfer

Gene transfer can occur through two main types⁹:

• Vertical gene transfer is the transfer of genetic material from parent to offspring cells.
• Horizontal gene transfer is the transfer to another recipient cell that is not in a parent-offspring relation¹⁰.

Vertical gene transfer

Vertical gene transfer is a vital mechanism of genetic inheritance, where DNA is passed from parents to offspring. This foundational concept in genetics involves the transmission of genetic material, with half of the DNA contributed by the mother and the other half by the father¹¹.

Vertical gene transfer can involve two types of reproduction:

• Sexual reproduction – a fusion of the male (sperm) and female (egg) gametes to form a zygote through a process called fertilization.
• Asexual reproduction – continuous cell division where a single parent transfers its genetic material to the offspring without the involvement of gametes. Example – Binary fission in bacteria. Here, genetically identical clones are formed¹². This type of vertical gene transfer is seen in prokaryotes and some eukaryotes, like paramecium¹³.

Gene transfer in higher eukaryotes can be applied therapeutically through somatic gene therapy, which involves manipulating non-reproductive somatic cells in the body, or germline gene therapy, which alters genetic information in germ cells¹⁴.

Biological significance: Key to the passing of traits across generations

Vertical gene transfer plays a vital role in maintaining species continuity and genetic integrity. It is responsible for transmitting inherited traits that include physical characteristics and genetic mutations¹⁵.

While vertical gene transfer preserves genetic stability in asexual organisms, it promotes genetic diversity in sexual reproduction, enabling populations to adapt and evolve. This process is fundamental to evolutionary theory, as it allows beneficial traits to persist and be shaped by natural selection over time.

Horizontal (lateral) gene transfer

Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is defined as the movement of genetic material between different organisms, without involving parents and offspring. It enables immediate or delayed effects in the recipient¹⁶. HGT is the nonsexual transfer of genes between organisms, playing a major role in evolution. While HGT is most frequent among bacteria and Archaea, it also occurs across diverse species¹⁷.GT can occur between:

Prokaryote to prokaryote: Rhizobial symbiosis genes are frequently located on plasmids or symbiotic islands that can be horizontally transferred between bacterial species. These genes often show phylogenies that differ from their host’s core genome, indicating independent evolutionary histories¹⁸.

Eukaryote to eukaryote:

• Organelle to organelle: A well-known historical example is the transfer of mitochondrial and chloroplast DNA to the nucleus in early eukaryotes¹⁹.
• Plant to plant: Another notable example of horizontal gene transfer is the integration of tumor-inducing (T-DNA) genes from Agrobacterium species into host plant genomes. These genes, found in species like Nicotiana, Linaria, and Ipomoea, are maintained in the germline and may contribute to plant evolution without causing disease²⁰.

Prokaryote to eukaryote:

For example, Wolbachia, a bacterium, has been observed to exhibit HGT in various insects, including aphids, mosquitoes, beetles, fruit flies, and wasps. In one case, about 30% of a Wolbachia genome was found integrated into a beetle’s X-chromosome, with roughly half of the transferred genes being transcribed at low levels, though the biological significance of this transcription remains unclear¹⁹.

Tardigrades, or water bears, are microscopic animals known for surviving extreme environmental conditions. In 2015, researchers discovered that about 17.5% of their genes come from foreign sources like bacteria, fungi, plants, and archaea—nearly double the previously known maximum for horizontal gene transfer (HGT). These foreign genes are thought to enhance the tardigrade’s stress resistance, similar to how bacteria endure harsh environments¹⁶.

Importance in evolution

Transposable elements (TEs), such as LINEs (L1), are key mediators of horizontal gene transfer (HGT) and play significant roles in human genome evolution, disease development, and genomic instability¹⁶. For example, HGT is common in the gut microbiome, driving microbial evolution through mechanisms like transduction and conjugation. Several bioinformatic tools help track antibiotic resistance genes and quantify HGT’s impact on the gut²¹.

While most TEs can no longer move, they still contribute to chromosomal recombination and mutations. TEs fall into two main classes, retrotransposons (class I) that move via an RNA intermediate and DNA transposons (class II) that use a cut-and-paste method, with many non-autonomous elements like SINEs relying on enzymes from autonomous TEs for mobility²².

They are implicated in different diseases and disorders by disrupting gene regulation and promoting genomic instability:

• Cancer: TE becomes more active due to weakened suppression mechanisms and mobilizing through RNA intermediates²³.
• Genetic disorder: An Alu (a Transposon) insertion in an intron of the F8 gene causes aberrant splicing, leading to hemophilia A due to exon skipping²².
• Metabolic disorder: A study found that LINE-1 insertion in exon 7 of LDLR causes familial hypercholesterolemia²⁴.
• Neurological disorders: Epigenetic changes during development and aging can activate retrotransposons like LINE-1, disrupting brain gene function and contributing to psychiatric and neurodegenerative disorders²⁵.
• Neurogenerative diseases: In neurodegenerative diseases like Alzheimer’s and Parkinson’s, age-related loss of TE repression may trigger their activation, causing DNA damage, gene disruption, and neuroinflammation²⁶.

HGT has contributed to the evolution of thermophily, enabling organisms to survive high temperatures. A key example is the Cyanidiophyceae, including Galdieria and Cyanidioschyzon, which evolved thermophily around a billion years ago²⁷. In land plants, which evolved from charophyte algae, complex interactions with microbes like rhizobacteria and fungi may have facilitated gene transfer²⁸.

Cross-species transfer

Cross-species transfer refers to a process in which the genetic material is transformed from one species to another. This innovative approach helps in understanding genetic engineering, evolutionary biology, and synthetic biology to study and manipulate complex biological systems²⁹.

Cross-species genome transfer has significantly advanced, with researchers successfully transferring whole genomes between different organisms. For example, the genome of Synechocystis PCC6803 was transferred into Bacillus subtilis, resulting in a chimeric chromosome, and B. subtilis was further developed as a platform for genome transfer methods.

Advances in platforms like Saccharomyces cerevisiae and efforts to clone and transfer large DNA fragments in Escherichia coli are expanding the possibilities for cross-species genome transfer³⁰.

Horizontal gene transfer in bacteria

HGT is a key driver of evolution in prokaryotes, allowing organisms to gain novel genes from a global gene pool to adapt to new niches, such as acquiring antibiotic resistance or virulence factors. HGT has been observed in insects, nematodes, and land plants, particularly involving mitochondrial genes, but it can be rare due to reproductive barriers³¹. Genes derived via HGT have also contributed to the evolution and adaptation of green plants with several metabolic and morphological innovations³².

In bacteria, HGT largely occurs through mobile genetic elements (MGEs) like bacteriophages, plasmids, and integrative and conjugative elements (ICEs)³³. Bacteriophages primarily propagate within species due to host genetic similarity, while plasmids and ICEs can cross species boundaries, making them key drivers of interspecies HGT. For example, the ICE present in Acinetobacter baylyi boosts cross-species HGT by killing nearby bacteria (bacterial predation) and making it resistant to antibiotics. This can combat the rise of multidrug-resistant superbugs³⁴.

The bacteria often acquire resistance by horizontal gene transfer mediated by mobile genetic elements such as insertion sequences, transposons, plasmids, integrative conjugative elements, and integrons carrying gene cassettes. Common gene cassettes include those encoding resistance to antibiotics like aadA (aminoglycoside resistance), and dfrA (trimethoprim resistance), which are frequently found in ESKAPEE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa,  Enterobacter  spp., and Escherichia coli) pathogens—the most problematic hospital-acquired bacteria³⁵. For example, staphylococcal cassette chromosome (SCC) elements are complex mobile genetic elements that frequently carry antimicrobial resistance and, in some cases, virulence genes³⁷.

Mechanisms of gene transfer in bacteria

The three classical mechanisms for HGT in prokaryotes are transformation, conjugation, and transduction. While conjugation and transformation are the primary contributors to bacterial HGT, transduction can be generalized and specialized according to the type of gene transfer. Bacteriophages, which are abundant and diverse, often carry virulence factors and contribute to bacterial strain variation through prophage sequences³⁸.

HGT can benefit cells by introducing useful functions, such as a selective survival advantage to the host, as seen in ICEs of Streptococcus salivarius^39,40. However, it can be harmful if the acquired genes are incompatible or selfishly replicating elements.

In early prokaryotic evolution, HGT rates were likely very high, blurring the distinction between lineages. However, as HGT rates decrease, distinct lineages with unique gene sets begin to emerge, leading to tree-like evolutionary patterns. Researchers found that earlier gene transfers are now widespread, and gene exchange is more common among co-occurring, abundant, or interacting species. Host-associated specialists mostly exchange genes with each other, while generalists exchange genes consistently across habitats⁴¹. Below are listed some of the benefits of bacterial horizontal gene transfer:

Antibiotic resistance

Antibiotic resistance poses a significant global health threat. The seriousness of this issue is highlighted by multi-drug resistant (MDR) bacterial infections causing around 700.000 deaths annually worldwide, and this number could increase to 10 million in 2050⁴². HGT via plasmid-mediated conjugation plays an important role in spreading resistance genes. This process enables DNA transfer across genera, phyla, and domains, forming primary transconjugants after initial donor conjugation. An example is the spread of β-lactam antibiotic resistance through conjugation, with the plasmid pCT spreading the blaCTX-M-14 across continents to animals and humans. Regarding transformation, although direct evidence is yet to be obtained, several clinical pathogens are known to undergo natural transformation like Streptococcus. Further, the role of transduction has also been outlined indirectly in the spread of antibiotic resistance, as seen in experiments with S. aureus hospital isolates⁴³.

However, the spread of antibiotic resistance genes (ARGs) involves both HGT and VGT. The inhibitory mechanisms of antibiotics influence HGT, while VGT plays a significant role in driving ARG propagation within bacterial communities¹⁵.

Interestingly, antibiotics have been shown to increase the rate of conjugation, as seen by experiments with stimulated conjugation in S. aureus after vancomycin treatment. Antibiotics such as trimethoprim can enhance both HGT and VGT rates, depending on their effects on donors, recipients, and transconjugants. Such findings provide details about the dynamics of resistance spread in environmental settings⁴⁴.

Evolutionary significance

HGT plays an essential role in the evolution of bacteria, with evidence found in many genomes. Although not all transfer events are biologically significant, adaptive transfers can produce detectable genetic signatures, such as gene-specific sweeps or increased transfer rates for ecologically relevant traits.

Various mechanisms of HGT, its genetic and evolutionary impacts, and methods to detect adaptive transfers highlight its role in facilitating adaptation to new environments⁴⁵.

Comparing horizontal and vertical gene transfer

Both horizontal and vertical gene transfer are essential biological processes in prokaryotes and eukaryotes. VGT ensures the faithful transmission of essential genetic traits, preserving species integrity over generations through sexual or asexual transfer of genes. Although slower than HGT, VGT allows for the accumulation of beneficial mutations and the gradual refinement of traits⁴⁶.

However, their mechanisms and evolutionary patterns differ, resulting in distinct functional outcomes. Below are some of the key differences between them^9,17,46,47.

Key differences

Methods of gene transfer

Gene transfer technologies enhance the efficiency of genetic manipulation in living organisms. These methods are categorized into natural processes, such as conjugation, transposition, bacterial transformation, and viral transduction, as well as artificial techniques that physically transfer genes, like biolistic transformation, microinjection, and protoplast fusion. Both approaches enable precise gene alterations across different organisms. Selecting the appropriate DNA transfer method is vital for successful transfections, with factors like target cell type, transfection endurance, and gene selection playing key roles¹.

Natural methods

Three major natural methods of bacterial gene transfer are known: conjugation, bacterial transformation, and transduction. Each method has its own applications and considerations, for example, for expressing an enzyme, expressing the recombinant DNA, recombinant enzyme production, identification of transformed cells, and organism regeneration such as restoring wild-type genotypes of bacteria through chromosomal curing in transformation-mediated stress adaptation⁴⁸.

Conjugation

In bacterial conjugation, genetic material is transferred between bacterial cells via direct contact or bridge-like pili connections. This process is common in prokaryotes and is frequently used in research for creating new strains⁴⁹. Conjugal transfer of plasmids has been observed in lactic acid bacteria (LAB), with improved Lactococcus strains available in the market for many years. For example, LAB can transfer the pediocin PA-1-like bacteriocin gene via conjugation, with transfer varying by strain and conditions. The results showed that Pediococcus acidilactici transferred the gene both in vitro and in situ, Enterococcus faecium only in situ, and Lactobacillus plantarum showed no transfer. This natural gene spread among LAB may aid dairy fermentation and culture development⁵⁰.

The transfer of DNA (transferred DNA or T-DNA) seen in Agrobacterium tumefaciens uses a bacterial type IV secretion system (VirB/VirD4) to transfer T-DNA to host plants⁵¹. This system originated from bacterial conjugal systems and is used in genetic engineering for developing transgenic plants⁵². Developing these plants can aid in pest resistance, tolerance to biotic and abiotic stress, enhanced yield, and nutritional benefits⁵³. The most famous example is the Bacillus thuringenesis or Bt crops; many cereals and fruit crops are other examples¹²⁰.

Transformation

Transformation is a process where bacterial cells take up DNA from their environment and integrate it into their genome, either through homologous genetic recombination or by retaining plasmids⁴⁸.

The ability to take up exogenous DNA varies between bacterial species and depends on the cell’s physiological state and environmental conditions. While some species, like Bacillus subtilis and Streptococcus pneumoniae, are naturally competent, others, such as S. thermophilus, require specific growth conditions to exhibit competence⁵⁴. The calcium chloride treatment is often used in labs to induce competence in E. coli, followed by the heat shock-based transformation⁵⁵. However, many species still lack demonstrated natural competence despite possessing the necessary genes, such as Flavobacterium johnsoniae⁵⁶.

Transduction

Transduction is a process where bacteriophages transfer DNA between bacteria, allowing horizontal gene transfer through viral infection. Both the donor and recipient bacteria must be susceptible to the same bacteriophage for the transfer to occur. The process involves the reproduction of the phage in the donor and its subsequent infection of the recipient cells. It can also involve retroviruses, which are single-stranded positive-sense RNA viruses¹.

The genetic material is transferred between bacteria without requiring direct cell-to-cell contact. It includes two types:

• Generalized transduction, which transfers random DNA fragments.
• Specialized transduction, which transfers specific DNA regions.

While transduction has been used to study genetic properties in bacteria like Lactococcus and Lactobacillus, with studies also suggesting that transduction is the cause of antibiotic resistance spread and establishment of S. aureus as a pathogen. Efforts are also made to eliminate undesirable genetic material, such as antibiotic-resistance genes or harmful metabolite-producing genes, through methods like DNA plasmid delivery or mutagenesis^57,58.

Artificial methods

Advancements in gene transfer technologies have enabled the genetic manipulation of higher organisms in addition to their use in bacteria. Artificial DNA transfer methods are categorized into physical, chemical, and electrical techniques, each with distinct applications. These classical methods are widely used in gene transfer¹.

Electroporation

Electroporation is an efficient method for gene delivery that creates temporary pores in cell membranes using electrical pulses to allow molecules to enter the cytoplasm. Major advantages include the reproducibility of gene expression, the ability to target many cell/tissue types, and the potential for in vivo delivery⁵⁹.

This technique is widely used in biotechnology for electrotransformation, inactivation, extraction, and biomass drying. It is particularly effective for in vivo gene transfer applications, such as delivering plasmid DNA to muscle and tumor tissues⁶⁰. It has shown promise in preclinical therapeutic studies, for example, the delivery of interleukin-12 (IL-12) to drive antitumor responses in a mouse model of melanoma and studies reporting the promise of electroporation for intradermal delivery of vaccines^61,62

The method’s efficiency can be improved by optimizing several parameters like field homogeneity, cell size, and electrode positioning to reduce cell damage and enhance gene transfer¹.

Calcium phosphate precipitation

Calcium phosphate transfection is a less commonly used and older non-viral method for introducing foreign DNA into cells, involving DNA encapsulation, endocytosis, and expression. It is a chemical method of gene transfer that forms calcium phosphate-DNA complexes to promote DNA uptake by cells⁶³.

The calcium phosphate precipitation method effectively transfects high cell-density cultures, which is essential for achieving high titers⁶⁴. An example is the use of this approach to obtain high titers of lentiviral vectors to transduce primary human T lymphocytes and generate chimeric antigen receptor T cells. This makes it a suitable technique for applications requiring large-scale gene expression regulation⁶⁵.

It is popular for its simplicity and low cost, but can be limited by cellular toxicity and inconsistent gene transfection methods. DNA uptake improves when delivered as a calcium phosphate–DNA coprecipitate, entering cells via endocytosis. Efficiency increases with steps like glycerol shock and chloroquine treatment. The protocol, originally optimized for CHO and HEK 293 cells, is easily adapted for both adherent and nonadherent cell types⁶⁶.

Calcium phosphate nanoparticles also promise in drug delivery systems due to their biocompatibility, biodegradability, and strong binding affinity for nucleic acids and therapeutic drugs. Recent developments focus on designing stable, targeted, and pH-responsive nanocarriers for future clinical applications⁶⁷.

Microinjection

Microinjection is a precise method of gene transfer where DNA is directly injected into a cell’s nucleus using a fine needle. It is ideal for applications requiring high specificity, such as creating transgenic organisms, due to its controlled DNA delivery (the famous example Dolly was cloned using microinjection)^1,68.

It is a precise and versatile technique essential in biomedical research and healthcare, enabling the delivery or retrieval of biological materials with accuracy⁶⁹. Over the past century, it has been pivotal in advancements like cloning and transgenics, understanding mechanisms such as aiptasia microinjection (a model for coral-algae interaction), helping understand the molecular mechanisms of coral symbiosis, gene targeting, infertility treatment, as shown in a study in mice where microinjection of adenoviruses carrying Kitl restored fertility in female mice^70,71. The use of microinjection in genome editing, including the use of the CRISPR-Cas9 system was reported in a study where both techniques successfully edited wheat microspores⁷². However, it is labor-intensive and demands specialized equipment and expertise.

With innovations like computerized instrumentation, such as the “autoinjector” robot microinjector and AI-driven nanomanipulators, microinjection is poised to remain a vital tool in both systemic and precision approaches to research and medicine^73,74. For example, an automated micropipette-based microinjection system has been developed to achieve precise single-cell transfection by controlling injection pressure and time. This technology enables quantitative delivery of materials into individual cells⁷⁵.

Liposome-mediated transfer

Liposomes are artificial vesicles made of phospholipids and cholesterol, containing an aqueous solution enclosed in a lipid bilayer. It is also used as a transfection agent to deliver materials like nucleic acids and proteins to cells. The transfection reagent Lipofectamine has been effective in improving siRNA uptake in nematodes⁷⁶.

Liposome-mediated transfer, or lipofection, uses liposomes to encapsulate DNA and deliver it into cells, with cationic liposomes like 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and other additives like carboxymethyl-β-cyclodextrin for improving efficiency and minimizing toxicity. This versatile method can deliver many genetic materials, including DNA, RNA, proteins, and peptides. However, challenges with reproducibility and efficiency remain a limitation^77,78.

Clinically, liposomes have been used as drug delivery systems, such as the first FDA approved nano-drug, doxorubicin delivered using PEGylated liposomes and for therapeutics, such as targeting ovarian cancer in mice models and vaccine development, for example, the use of liposomal DNA delivery elicited immunity in mice against three malarial antigens, the Circumsporozoite protein, the C terminus of merozoite surface protein 1 from Plasmodium vivax and Plasmodium falciparum Rhoptry antigen 5^79,80,81,82. With recent genetic advances, liposome nanoparticles are now also being explored for gene delivery, expanding their applications in biotechnology and medicine.

Gene gun-mediated transfer

The gene gun method uses accelerators to deliver particles coated with exogenous genes into cells, enabling gene integration and expression. It is widely used in crops and fruit trees like wheat, corn, beans, and citrus. Gene gun technology also shows strong potential for genetic research in forest trees⁸³.

It is also used to investigate the plant immune responses. For example, gene gun-mediated transfer is effectively used in barley to study resistance against powdery mildew by transiently expressing Mlo genes in leaf epidermal cells. This approach allows rapid functional analysis without creating stable transgenic lines⁸⁴.

Gene gun-mediated transformation is successful in cells with strong regeneration and physiological activity⁸³. For example, a study demonstrated that transferring the bone morphogenetic protein 2 (BMP-2) gene using a gene gun significantly enhanced bone healing in a rabbit fracture model by increasing BMP-2 expression and promoting bone callus formation⁸⁵.

Viruses are powerful tools for gene therapy, vaccines, and cancer treatment due to their gene transfer ability. Common viral vectors like adenoviruses and retroviruses are being optimized to improve their safety and effectiveness, with some preclinical trials showing promising vaccine results. The future of cancer therapy may rely on the oncolytic properties of viruses, offering treatments without the harmful side effects of traditional methods⁸⁶.

Gene transfer agents (GTAs), recently identified in several species, originate from ancient bacteriophages but have evolved unique features distinct from typical viruses. Their regulation is complex, involving quorum sensing, stress responses, and various regulatory proteins. Unlike typical horizontal gene transfer mechanisms, GTA release involves the lysis of a significant portion of the producing cell population, posing an evolutionary cost such as fitness reduction⁸⁷.

Applications of gene transfer

Gene transfer has a myriad of applications that extend from treating genetic diseases to creating genetically modified organisms. The purpose of gene transfer entails in creation of disease-resistant and crops with higher yields, as seen in improved wheat yield and water use carrying the sunflower gene HaHB4^88,89, the production of therapeutic primary antibodies like Anti-Prostaglandin E Synthase/MPGES-1 antibody and personalized medicines.

Biotechnology: Use in developing products and processes

Gene transfer is increasingly used in biotechnology to engineer the immune system through in vivo gene transfer and ex vivo gene transfer methods⁹⁰. Nucleic acids such as plasmid DNA (pDNA), mRNA, microRNA (miRNA), molecules like small interfering RNA (siRNA), and immunostimulatory nucleic acids used in immunotherapy, such as the promise of CpG oligodeoxynucleotides for cancer are delivered to overexpress genes, knock down gene expression, or elicit immune responses, each with unique properties and intracellular activity⁹¹. This nucleic acid delivery can also activate the innate immune system via pathways like Toll-like receptors (TLRs), with potential for engineering to optimize their effects.

Gene therapy is important in producing genetically modified (GM) plants through recombinant DNA technology as a solution to the growing population and climate change^92,93. This process introduces beneficial traits like enhanced nutrition, pest resistance, and drought tolerance. The advent of next generation sequencing (NGS) and CRISPR technology helps in evaluating the risks of gene transfer to other organisms. Agrobacterium-mediated transformation and gene gun-mediated microparticle bombardment are the two main methods for plant genetic modification, with Agrobacterium traditionally used in dicots and gene gun widely applied in various plants^94,95.

Advances in technologies like MetaCHIP allow for reference-independent detection of HGT in metagenomic datasets, enabling the study of microbial community dynamics. This tool can identify both recent and older HGTs, offering valuable insights into gene transfer related to antibiotic resistance and microbial evolution. MetaCHIP enhances our understanding of the role of HGT in microbial ecology⁹⁶.

Medicine

Transfer of genes in gene therapy has advanced significantly, with a growing number of approved products and promising developments in clinical trials. The evolution of strategies, including vectors, target cells, transferred genes, and ex-vivo/in-vivo methods, highlights the expanding applications, particularly in cancer. For example, Fomivirsen is the first approved gene therapy for the treatment of cytomegalovirus retinitis in immunocompromised patients, and ciltacabtagene autoleucel was approved in 2022 for relapsed multiple myeloma⁹⁷.

Trends in approaches and innovations from pioneering countries provide valuable direction for future clinical and therapeutic developments. Further, it is also helpful for treating viral infections and regenerative medicines, such as prostaglandin-e-synthase antibody used in rheumatoid arthritis, osteoarthritis, and inflammatory myopathies⁹⁸.

Gene transfer is also used to create chimeric antigen receptor (CAR) T-cells, which are genetically engineered to specifically attack cancer cells, such as those expressing CD19 and GCC in metastatic colorectal cancer^99,100. Innovations in CAR T-cell strategies, including dual-targeting antigens and combining with immune checkpoint inhibitors, are being explored to improve outcomes. Gene delivery is essential for advances in gene therapy and biotechnology, with recent improvements in vectors and techniques enhancing efficiency and safety. Emerging trends include integrating nanotechnology and CRISPR-Cas systems, as well as optimizing delivery methods¹⁰¹.

Clinical research

Gene transfer advances medical research by introducing specific genes into model organisms or cultured cells, helping researchers study disease mechanisms like Alzheimer’s and cancer metastasis¹⁶.

It enables the creation of transgenic animal models, such as mice with human amyloid-beta genes, to better understand human diseases. Genetically engineered mice created through gene transfer technologies have revolutionized the study of human diseases by enabling insights into molecular mechanisms, cellular pathways, and gene expression profiles linked to disease development. These models play a critical role in advancing translational research and developing therapeutic strategies to improve patient care¹⁰².

Gene transfer is also vital in drug development, allowing the production of therapeutic proteins like insulin and the validation of potential drug targets¹⁰³. Additionally, gene transfer has been used in vaccine development, for example, the mRNA COVID-19 vaccine, and improved drug delivery systems using nanoparticles and viral vectors¹⁰⁴. Gene transfer has also been studied for bioremediation, where the biodegradation potential is stimulated by facilitating HGT between indigenous microbes at a site and plasmids¹⁰⁵.

Ethical considerations and challenges

Gene transfer faces significant ethical and social challenges. Ethical concerns arise over the potential misuse of the technology for non-therapeutic purposes, such as genetic enhancement or altering physical traits, which creates hurdles in the responsible application of gene manipulation^106,107. These issues complicate the broader acceptance and regulation of gene transfer technologies. Gene therapy research faces major criticisms, including its irreversible effects, potential impact on the gene pool, consent challenges, and risk of harm. The United States established the recombinant DNA advisory committee (RDAC) to oversee human gene therapy research, promote safety, and ensure transparency through public reporting and protocol review¹⁰⁸. Ethics committees must ensure gene transfer trials provide valuable scientific and social benefits, as participants face serious risks. With no gene therapies commercialized yet, trials should prioritize gathering toxicity data and long-term follow-up to maximize social value¹⁰⁷.

Regulatory concerns

Gene transfer faces regulatory challenges^{109,110,111,112} like:

• Unintended genetic modifications, such as off-target effects and mutations in the genome.
• Germline alterations with potential long-term consequences, as this can be transmitted from generation to generation.
• Viral gene transfer methods can cause an immune response in the body.
• Prenatal and in-utero studies cause significant risks as the defects can be transferred to the fetus. Hence, this process needs careful evaluation.

Stringent oversight by the regulatory bodies like RDAC, institutional review boards (IRBs), institutional biosafety committees (IBCs), and the Food and Drug Administration (FDA) is essential during the transition from preclinical to clinical research to ensure safety and ethical compliance ¹¹³.

Certain precautions should be followed to design and produce gene therapy products (GTP). These precautions include choosing the appropriate delivery vector based on the target tissue and designing the expression cassette to ensure clinically relevant gene expression levels. Additionally, gene expression must be highly specific to prevent unwanted side effects and off-target impacts¹¹⁴.

Public perception and safety

Public perception of gene transfer is shaped by ethical concerns, including fears of genetic enhancement and altering traits beyond therapeutic needs, as well as its impact on cultural and social identity¹⁰⁶.

The perception of gene transfer and gene editing varies greatly. An example is an opinion-based survey conducted in the US, which found more safety results in the agricultural field than in the medical field. A lack of public understanding and dialogue about the safety and benefits of gene editing has contributed to skepticism despite extensive scientific evidence supporting its safety¹¹⁵.

Early gene transfer research raised significant ethical, social, and technical concerns, including fears about germline modification, environmental risks, and the long-term effects of altering the human genome. Limited scientific understanding, particularly of immune responses and lifelong exposure, has sparked debate over the readiness of gene transfer for clinical trials, emphasizing the need for stricter oversight¹¹⁶. Proactive communication strategies are essential to bridge the gap between public opinion and scientific consensus, as seen with greater acceptance of therapeutic uses for diseases compared to cosmetic or non-essential applications.

FAQs

How do viral vectors work in gene transfer?

Viral vectors are modified viruses used to deliver genetic material into cells for gene transfer. They work by infecting target cells and introducing the desired gene into the cell’s genome. These vectors are highly effective for gene transfer, allowing targeted modification of specific cell types or tissues and the expression of therapeutic genes. Various virus types, including adenoviruses, retroviruses, and poxviruses, among others, are being explored for delivering genes with either transient or permanent expression. The selection of a viral vector for clinical use depends on factors like transgene expression efficiency, production ease, safety, toxicity, and stability.

What are the long-term effects of gene therapy?

The long-term effects of gene therapy can vary depending on the specific treatment, the type of disease, and the method of delivery. In some cases, patients experience sustained improvements in symptoms or even cures. In contrast, may face complications such as immune reactions, insertional mutagenesis, or the loss of therapeutic genes over time. Ongoing research aims to better understand and mitigate these risks to ensure the safety and efficacy of gene therapies in the long run as seen in a study including 10 patients who got corrected stem cells for adenosine deaminase (ADA) where gene therapy effects remained stable for up to 11 years, but outcomes varied depending on the amount of engrafted, gene-corrected stem cells. Higher levels of corrected cells generally led to better results^117,118,119.

What is the difference between in vivo and ex vivo gene therapy?

In vivo gene therapy involves the direct delivery of therapeutic genes into a patient’s body, typically through injections or viral vectors, where the gene modifies the cells within the body. Ex vivo gene therapy, on the other hand, involves removing cells from the patient, modifying them outside the body (in the lab), and then transplanting the modified cells back into the patient. The key difference lies in where the gene modification occurs—inside the body (in vivo) or outside the body (ex vivo).

References

1. Ozyigit, I.I. Gene transfer to plants by electroporation: methods and applications. Molecular Biology Reports. 47 (4), 3195–3210 (2020).
2. Moses, P. Gene transfer methods applicable to agricultural organisms. National Academies Press (US). (2016).
3. Sun, D., Jeannot, K., Xiao, Y., et al. Horizontal gene transfer mediated bacterial antibiotic resistance. Frontiers in Microbiology. 10 (2019).
4. Mahdizade Ari, M., Dadgar, L., Elahi, Z., et al. Genetically engineered microorganisms and their impact on human health. International Journal of Clinical Practice. 1–38 (2024).
5. Gene Therapy Program. FDA-approved gene therapies. Boston Children’s Hospital.
6. Khan, S., Ullah, M.W., Siddique, R., et al. Role of recombinant DNA technology to improve life. International Journal of Genomics. 2016 (2405954), 1–14 (2016).
7. Khobragade, A., Bhate, S., Ramaiah, V., et al. Efficacy, safety, and immunogenicity of the DNA SARS-CoV-2 vaccine (ZyCoV-D): the interim efficacy results of a phase 3, randomised, double-blind, placebo-controlled study in India. The Lancet. 399 (10332), 1313–1321 (2022).
8. Rafeeq, H., Afsheen, N., Rafique, S., et al. Genetically engineered microorganisms for environmental remediation. Chemosphere. 310, 136751 (2023).
9. Bethke, J.H., Ma, H.R., Tsoi, R., et al. Vertical and horizontal gene transfer tradeoffs direct plasmid fitness. Molecular Systems Biology. 19 (2), (2022).
10. Soucy, S.M., Huang, J., Gogarten, J.P. Horizontal gene transfer: building the web of life. Nature Reviews Genetics. 16 (8), 472–482 (2015).
11. David, I., Canario, L., Combes, S., et al. Intergenerational transmission of characters through genetics, epigenetics, microbiota, and learning in livestock. Frontiers in Genetics. 10 (2019).
12. Harpring, M., Cox, J.V. Plasticity in the cell division processes of obligate intracellular bacteria. Frontiers in Cellular and Infection Microbiology. 13, 1205488 (2023).
13. Long, H., Johri, P., Goût, J.-F., et al. Paramecium genetics, genomics, and evolution. Annual Review of Genetics. 57 (1), 391–410 (2023).
14. Smith, K.R. Gene transfer in higher animals: theoretical considerations and key concepts. Journal of Biotechnology. 99 (1), 1–22 (2002).
15. 1Li, B., Qiu, Y., Song, Y., et al. Dissecting horizontal and vertical gene transfer of antibiotic resistance plasmid in bacterial community using microfluidics. Environment International. 131, 105007 (2019).
16. Emamalipour, M., Seidi, K., Zununi Vahed, S., et al. Horizontal gene transfer: from evolutionary flexibility to disease progression. Frontiers in Cell and Developmental Biology. 8 (229), (2020).
17. Islam, T., Azad, R.B., Kasfy, S.H., et al. Horizontal gene transfer from plant to whitefly. Trends in Biotechnology. 41 (7), 853–856 (2023).
18. Andrews, M., De Meyer, S., James, E., et al. Horizontal transfer of symbiosis genes within and between rhizobial genera: occurrence and importance. Genes. 9 (7), 321 (2018).
19. Dunning Hotopp, J.C. Horizontal gene transfer between bacteria and animals. Trends in Genetics. 27 (4), 157–163 (2011).
20. Quispe-Huamanquispe, D.G., Gheysen, G., Kreuze, J.F. Horizontal gene transfer contributes to plant evolution: the case of Agrobacterium T-DNAs. Frontiers in Plant Science. 8 (2017).
21. McInnes, R.S., McCallum, G.E., Lamberte, L.E., et al. Horizontal transfer of antibiotic resistance genes in the human gut microbiome. Current Opinion in Microbiology. 53, 35–43 (2020).
22. Chénais, B. Transposable elements and human diseases: mechanisms and implication in the response to environmental pollutants. International Journal of Molecular Sciences. 23 (5), 2551 (2022).
23. Lee, E., Iskow, R., Yang, L., et al. Landscape of somatic retrotransposition in human cancers. Science. 337 (6097), 967–971 (2012).
24. Song, Y., Abdel, R., Omar, O.M., et al. A case report of an Egyptian family with familial hypercholesterolemia and an exonic LINE‐1 insertion in LDLR. Molecular Genetics & Genomic Medicine. 12 (3), (2024).
25. Jönsson, M.E., Garza, R., Johansson, P.A., et al. Transposable elements: a common feature of neurodevelopmental and neurodegenerative disorders. Trends in Genetics. 36 (8), 610–623 (2020).
26. Ravel‐Godreuil, C., Znaidi, R., Bonnifet, T., et al. Transposable elements as new players in neurodegenerative diseases. FEBS Letters. 595 (22), 2733–2755 (2021).
27. van Etten, J., Bhattacharya, D. Horizontal gene transfer in eukaryotes: not if, but how much? Trends in Genetics. 36 (12), 915–925 (2020).
28. Ma, J., Wang, S., Zhu, X., et al. Major episodes of horizontal gene transfer drove the evolution of land plants. Molecular Plant. 15 (3), (2022).
29. Yuan, H., Mancuso, C.A., Johnson, K., et al. Computational strategies for cross-species knowledge transfer and translational biomedicine. arXiv. arXiv:2408.08503v1 (2024).
30. Zhu, M.C., Cui, Y.Z., Wang, J.Y., et al. Cross-species microbial genome transfer: a review. Frontiers in Bioengineering and Biotechnology. 11 (2023).
31. Yue, J., Hu, X., Huang, J. Horizontal gene transfer in the innovation and adaptation of land plants. Plant Signaling & Behavior. 8 (5), e24130 (2013).
32. Wang, H., Li, Y., Zhang, Z., et al. Horizontal gene transfer: driving the evolution and adaptation of plants. Journal of Integrative Plant Biology. 65 (3), 613–616 (2023).
33. Redondo-Salvo, S., Fernández-López, R., Ruiz, R., et al. Pathways for horizontal gene transfer in bacteria revealed by a global map of their plasmids. Nature Communications. 11 (1), (2020).
34. Li, L., Liu, Y., Xiao, Q., et al. Dissecting the HGT network of carbon metabolic genes in soil-borne microbiota. Frontiers in Microbiology. 14 (2023).
35. Partridge, S.R., Kwong, S.M., Firth, N., et al. Mobile genetic elements associated with antimicrobial resistance. Clinical Microbiology Reviews. 31 (4), (2018).
36. Lorestani, R.C., Akya, A., Elahi, A., et al. Gene cassettes of class I integron-associated with antimicrobial resistance in isolates of Citrobacter spp. with multidrug resistance. Iranian Journal of Microbiology. 10 (1), 22 (2018).
37. Shore, A.C., Coleman, D.C. Staphylococcal cassette chromosome mec: recent advances and new insights. International Journal of Medical Microbiology. 303 (6-7), 350–359 (2013).
38. Schneider, C.L. Bacteriophage-mediated horizontal gene transfer: Transduction. Bacteriophages. 151–92 (2021).
39. Vogan, A.A., Higgs, P.G. The advantages and disadvantages of horizontal gene transfer and the emergence of the first species. Biology Direct. 6, 1 (2011).
40. Dahmane, N., Libante, V., Charron-Bourgoin, F., et al. Diversity of integrative and conjugative elements of Streptococcus salivarius and their intra- and interspecies transfer. Applied and Environmental Microbiology. 83(13), e00337-17 (2017).
41. Dmitrijeva, M., Tackmann, J., Matias Rodrigues, J.F., et al. A global survey of prokaryotic genomes reveals the eco-evolutionary pressures driving horizontal gene transfer. Nature Ecology & Evolution. 1-13 (2024).
42. Serra-Burriel, M., Keys, M., Campillo-Artero, C., et al. Impact of multi-drug resistant bacteria on economic and clinical outcomes of healthcare-associated infections in adults: Systematic review and meta-analysis. PLoS ONE. 15(1), e0227139 (2020).
43. Lerminiaux, N.A., Cameron, A.D.S. Horizontal transfer of antibiotic resistance genes in clinical environments. Canadian Journal of Microbiology. 65(1), 34–44 (2019).
44. Wu, R., Fang, J., Yang, Y., et al. Transmission pathways and intrinsic mechanisms of antibiotic resistance genes in soil-plant systems: a review. Environmental Technology & Innovation, 103985 (2024).
45. Arnold, B.J., Huang, I-T., Hanage, W.P. Horizontal gene transfer and adaptive evolution in bacteria. Nature Reviews Microbiology. 20, (2021).
46. Lorenzo-Díaz, F., Fernández-López, C., Lurz, R., et al. Crosstalk between vertical and horizontal gene transfer: plasmid replication control by a conjugative relaxase. Nucleic Acids Research. 45(13), 7774–7785 (2017).
47. Liu, F., Luo, Y., Xu, T., et al. Current examining methods and mathematical models of horizontal transfer of antibiotic resistance genes in the environment. Frontiers in Microbiology. 15 (2024).
48. Carvalho, G., Fouchet, D., Danesh, G., et al. Bacterial transformation buffers environmental fluctuations through the reversible integration of mobile genetic elements. mBio. 11(2) (2020).
49. Iglesias, S.M., Li, F., Federica Briani, et al. Viral genome delivery across bacterial cell surfaces. Annual Review of Microbiology. 78(1), 125–145 (2024).
50. Devi, S.M., Halami, P.M. Conjugal transfer of bacteriocin plasmids from different genera of lactic acid bacteria into Enterococcus faecalis JH2-2. Annals of Microbiology. 63(4), 1611–1617 (2013).
51. Chen, I., Christie, P.J., Dubnau, D. The ins and outs of DNA transfer in bacteria. Science. 310(5753), 1456–1460 (2005).
52. Ohmine, Y., Kiyokawa, K., Yunoki, K., et al. Successful transfer of a model T-DNA plasmid to E. coli revealed its dependence on recipient RecA and the preference of VirD2 relaxase for eukaryotes rather than bacteria as recipients. Frontiers in Microbiology. 9 (2018).
53. National Research Council, National Academy of Sciences, et al. Examples of GM technology that would benefit world agriculture. National Academies Press (US) (2000).
54. Kilb, A., Burghard-Schrod, M., Holtrup, S., et al. Uptake of environmental DNA in Bacillus subtilis occurs all over the cell surface through a dynamic pilus structure. PLOS Genetics. 19(10), e1010696 (2023).
55. Chang, A.Y., Chau, V.W.Y., Landas, J.A., et al. Preparation of calcium competent Escherichia coli and heat-shock transformation. Undergraduate Journal of Experimental Microbiology and Immunology. 1, 22–25 (2017).
56. Huang, M., Liu, M., Huang, L., et al. The activation and limitation of the bacterial natural transformation system: The function in genome evolution and stability. Microbiological Research. 252, 126856 (2021).
57. Mugwanda, K., Hamese, S., Van Zyl Winschau, F., et al. Recent advances in genetic tools for engineering probiotic lactic acid bacteria. Bioscience Reports. 43(1) (2023).
58. Fillol-Salom, A., Alsaadi, A., de Sousa, J.A.M., et al. Bacteriophages benefit from generalized transduction. PLoS Pathogens. 15(7) (2019).
59. Young, J.L., Dean, D.A. Electroporation-mediated gene delivery. Advances in Genetics. 89, 49–88 (2015).
60. Heller, L.C., Ugen, K., Heller, R. Electroporation for targeted gene transfer. Expert Opinion on Drug Delivery. 2(2), 255–268 (2005).
61. Lampreht Tratar, U., Loiacono, L., Cemazar, M., et al. Gene electrotransfer of plasmid-encoding IL-12 recruits the M1 macrophages and antigen-presenting cells inducing the eradication of aggressive B16F10 murine melanoma. Mediators of Inflammation. 1–11 (2017).
62. Generotti, A., Contreras, R., Zounes, B., et al. Intradermal DNA vaccine delivery using vacuum-controlled, needle-free electroporation. Molecular Therapy Nucleic Acids. 34, 102070 (2023).
63. Kofron, M.D., Laurencin, C.T. Development of a calcium phosphate co-precipitate/poly(lactide-co-glycolide) DNA delivery system: release kinetics and cellular transfection studies. Biomaterials. 25(13), 2637–2643 (2004).
64. Keyer, V., Syzdykova, L., Ingirbay, B., et al. Non‐industrial production of therapeutic lentiviral vectors: How to provide vectors to academic CAR‐T. Biotechnology and Bioengineering. 121(10), 3252–3268 (2024).
65. Lo, C.W., Lin, T., Ueno, M., et al. Optimization and characterization of calcium phosphate transfection in mesenchymal stem cells. Tissue Engineering Part C: Methods. 25(9), 543–552 (2019).
66. Calcium phosphate–mediated transfection of eukaryotic cells. Nature Methods. 2(4), 319–320 (2005).
67. Qiu, C., Wu, Y., Guo, Q., et al. Preparation and application of calcium phosphate nanocarriers in drug delivery. Materials Today Bio. 17, 100501 (2022).
68. Muthaiyan Shanmugam, M., Manoj, H. Microinjection for single-cell analysis and therapy. Handbook of Single Cell Technologies. 1, 1–27 (2021).
69. Zhang, Y., Yu, L.C. Microinjection as a tool of mechanical delivery. Current Opinion in Biotechnology. 19(5), 506–510 (2008).
70. Jones, V.A.S., Bucher, M., Hambleton, E.A., et al. Microinjection to deliver protein, mRNA, and DNA into zygotes of the cnidarian endosymbiosis model Aiptasia sp. Scientific Reports. 8(1) (2018).
71. Kanatsu-Shinohara, M., Lee, J., Miyazaki, T., et al. Adenovirus-mediated gene delivery restores fertility in congenitally infertile female mice. The Journal of Reproduction and Development. 68(6), 369–376 (2022).
72. Szabała, B.M., Święcicka, M., Łyżnik, L.A. Microinjection of the CRISPR/Cas9 editing system through the germ pore of a wheat microspore induces mutations in the target Ms2 gene. Molecular Biology Reports. 51(1) (2024).
73. Xu, W. Microinjection and micromanipulation: a historical perspective. Methods in Molecular Biology (Clifton, NJ). 1874, 1–16 (2019).
74. Shull, G., Haffner, C., Huttner, W.B., et al. Robotic platform for microinjection into single cells in brain tissue. EMBO Reports. 20(10) (2019).
75. Chow, Y.T., Chen, S., Wang, R., et al. Single cell transfection through precise microinjection with quantitatively controlled injection volumes. Scientific Reports. 6(1), 24127 (2016).
76. Adams, S., Pathak, P., Shao, H., et al. Liposome-based transfection enhances RNAi and CRISPR-mediated mutagenesis in non-model nematode systems. Scientific Reports. 9(1), (2019).
77. Elsana, H., Temidayo, O.B., Carr-Wilkinson, J., et al. Evaluation of novel cationic gene based liposomes with cyclodextrin prepared by thin film hydration and microfluidic systems. Scientific Reports. 9(1), (2019).
78. Mihailescu, M., Worcester, D.L., Carroll, C.L., et al. DOTAP: Structure, hydration, and the counterion effect. Biophysical Journal. 122(6), 1086–93 (2023).
79. Tenchov, R., Bird, R., Curtze, A.E., et al. Lipid nanoparticles—From liposomes to mRNA vaccine delivery, a landscape of research diversity and advancement. ACS Nano. 15(11), (2021).
80. Sercombe, L., Veerati, T., Moheimani, F., et al. Advances and challenges of liposome assisted drug delivery. Frontiers in Pharmacology. 6, (2015).
81. Son, J. S., Chow, R., Kim, H. A., Lieu, T. A., Xiao, M., Kim, S., et al. Liposomal delivery of gene therapy for ovarian cancer: a systematic review. Reproductive Biology and Endocrinology. 21(1), (2023).
82. Fotoran, W. L., Santangelo, R., de Miranda, B. N. M., Irvine, D. J., & Wunderlich, G. DNA-loaded cationic liposomes efficiently function as a vaccine against malarial proteins. Molecular Therapy Methods & Clinical Development. 7, 1–10 (2017).
83. Yin, Y., Wang, C., Xiao, D., et al. Advances and perspectives of transgenic technology and biotechnological application in forest trees. Frontiers in Plant Science. 12, (2021).
84. Leissing, F., Reinstädler, A., Thieron, H., et al. Gene gun-mediated transient gene expression for functional studies in plant immunity. Methods in Molecular Biology. 63–77 (2022).
85. Li, W., Wei, H., Xia, C., et al. Gene gun transferring-bone morphogenetic protein 2 (BMP-2) gene enhanced bone fracture healing in rabbits. International Journal of Clinical and Experimental Medicine. 8(11), 19982 (2015).
86. bin Umair, M., Akusa, F.N., Kashif, H., et al. Viruses as tools in gene therapy, vaccine development, and cancer treatment. Archives of Virology. 167(6), 1387–1404 (2022).
87. Craske, M.W., Wilson, J.S., Fogg, P.C.M., et al. Gene transfer agents: structural and functional properties of domesticated viruses. Trends in Microbiology. 32(12), 1200–11 (2024).
88. Esse, H.P., Reuber, T.L., Does, D., et al. Genetic modification to improve disease resistance in crops. New Phytologist. 225(1), (2019).
89. González, F.G., Capella, M., Ribichich, K.F., et al. Field-grown transgenic wheat expressing the sunflower gene HaHB4 significantly outyields the wild type. Journal of Experimental Botany. 70(5), 1669–81 (2019).
90. Neshat, S.Y., Tzeng, S.Y., Green, J.J., et al. Gene delivery for immunoengineering. Current Opinion in Biotechnology. 66, 1–10 (2020).
91. Callmann, C.E., Cole, L.E., Kusmierz, C.D., et al. Tumor cell lysate-loaded immunostimulatory spherical nucleic acids as therapeutics for triple-negative breast cancer. Proceedings of the National Academy of Sciences of the United States of America. 117(30), 17543–50 (2020).
92. Philips, J.G., Martin-Avila, E., Robold, A.V., et al. Horizontal gene transfer from genetically modified plants - Regulatory considerations. Frontiers in Bioengineering and Biotechnology. 10, (2022).
93. Gan, W.C., Ling, P.K., et al. CRISPR/Cas9 in plant biotechnology: Applications and challenges. BioTechnologia. 103(1), 81–93 (2022).
94. Mao, Y., Botella, J.R., Liu, Y., et al. Gene editing in plants: progress and challenges. National Science Review. 6(3), 421–37 (2019).
95. Gao, C., Nielsen, K.K., et al. Comparison between Agrobacterium-mediated and direct gene transfer using the gene gun. Methods in Molecular Biology (Clifton, NJ). 940, 3–16 (2013).
96. Song, W., Wemheuer, B., Zhang, S., et al. MetaCHIP: community-level horizontal gene transfer identification through the combination of best-match and phylogenetic approaches. Microbiome. 7(1), (2019).
97. Arabi, F., Mansouri, V., Ahmadbeigi, N., et al. Gene therapy clinical trials, where do we go? An overview. Biomedicine & Pharmacotherapy. 153(113324), (2022).
98. Korotkova, M., Jakobsson, P.-J., et al. Microsomal Prostaglandin E Synthase-1 in rheumatic diseases. Frontiers in Pharmacology. 1, (2011).
99. Jogalekar, M.P., Rajendran, R.L., Khan, F., et al. CAR T-cell-based gene therapy for cancers: new perspectives, challenges, and clinical developments. Frontiers in Immunology. 13, (2022).
100. Chen, N., Pu, C., Zhao, L., et al. Chimeric Antigen Receptor T Cells Targeting CD19 and GCC in Metastatic Colorectal Cancer. JAMA Oncology. (2024).
101. 1Quadir, S.S., Choudhary, D., Singh, S., et al. Genetic frontiers: Exploring the latest strategies in gene delivery. Journal of Drug Delivery Science and Technology. 101, 106316 (2024).
102. Doyle, A., McGarry, M.P., Lee, N.A., et al. The construction of transgenic and gene knockout/knockin mouse models of human disease. Transgenic Research. 21(2), 327–49 (2011).
103. Ebrahimi, S.B., Samanta, D., et al. Engineering protein-based therapeutics through structural and chemical design. Nature Communications. 14(1), 2411 (2023).
104. Fatima, M., Park, P.G., Hong, K.J., et al. Clinical advancements in mRNA vaccines against viral infections. Clinical Immunology. 271, 110424 (2024).
105. Ikuma, K., Gunsch, C.K., et al. Genetic bioaugmentation as an effective method for in situ bioremediation. Bioengineered. 3(4), 236–41 (2012).
106. 1Kimmelman, J., et al. The ethics of human gene transfer. Nature Reviews Genetics. 9(3), 239–44 (2008).
107. 1Kimmelman, J., et al. Recent developments in gene transfer: risk and ethics. BMJ. 330(7482), 79–82 (2005).
108. Feinberg, R.S., et al. Ethical issues in gene therapy research: An American perspective. ScienceDirect. 99–107 (2006).
109. Modrzejewski, D., Hartung, F., Lehnert, H., et al. Which factors affect the occurrence of off-target effects caused by the use of CRISPR/Cas: a systematic review in plants. Frontiers in Plant Science. 11, (2020).
110. Rubeis, G., Steger, F., et al. Risks and benefits of human germline genome editing: an ethical analysis. Asian Bioethics Review. 10(2), 133–41 (2018).
111. Shirley, J.L., de Jong, Y.P., Terhorst, C., et al. Immune responses to viral gene therapy vectors. Molecular Therapy. 28(3), 709–22 (2020).
112. Bose, S.K., Menon, P., Peranteau, W.H., et al. In utero gene therapy: progress and challenges. Trends in Molecular Medicine. 27(8), 728–30 (2021).
113. Lenzi, R.N., Altevogt, B.M., Gostin, L.O., et al. Oversight of gene transfer research. NIH.gov. National Academies Press (US). (2014).
114. Sivagourounadin, K., Ravichandran, M., Rajendran, P., et al. National guidelines for gene therapy product (2019): a road-map to gene therapy products development and clinical trials. Perspectives in Clinical Research. (2021).
115. McFadden, B.R., Rumble, J.N., Stofer, K.A., et al. U.S. public opinion about the safety of gene editing in the agriculture and medical fields and the amount of evidence needed to improve opinions. Frontiers in Bioengineering and Biotechnology. 12 (2024).
116. Lenzi, R.N., Altevogt, B.M., Gostin, L.O., et al. Gene transfer research: The evolution of the clinical science. National Academies Press (US). (2014).
117. Asher, D.R., Dai, D., Klimchak, A.C., et al. Paving the way for future gene therapies: a case study of scientific spillover from delandistrogene moxeparvovec. Molecular Therapy - Methods & Clinical Development. 30, 474–83 (2023).
118. Wu, C., Dunbar, C.E., et al. Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity. Frontiers of Medicine. 5(4), 356–71 (2011).
119. Reinhardt, B., Habib, O., Shaw, K.L., et al. Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency. Blood. 138(15), 1304–16 (2021).
120. Abbas, M.S.T. Genetically engineered (modified) crops (Bacillus thuringiensis crops) and the world controversy on their safety. Egypt J Biol Pest Control. 28, 52 (2018).