Transduction Efficiency Of Vectors Research Articles

<i>In vivo</i> evaluation of tropism and biodistribution of synthetic and natural adeno-associated viral vectors by next-generation sequencing

INTRODUCTION. The creation of synthetic adeno-associated virus (AAV) vectors during gene therapy development is a labour-intensive and expensive process. The optimal solution to minimise the time and costs associated with gene therapy development lies in the improvement of methods aimed at assessing AAV vector biodistribution and transduction efficiency in vivo.AIM. This study aimed to develop a new bioinformatics-based assessment method for synthetic AAV vector libraries to analyse AAV vector biodistribution and transduction efficiency in vivo.MATERIALS AND METHODS. The production of synthetic AAV vectors involved assigning AAV serotype-specific barcodes (12-nucleotide tags flanked at the 5' end with a sequence encoding the green fluorescent reporter protein). Plasmids carrying unique barcodes were propagated in competent Escherichia coli XL10-Gold cells and used to create two AAV libraries: L1 with a viral genome count of 1010 and L2 with a viral genome count of 1011. AAV production involved HEK293T cell transfection. L1 and L2 library vectors were administered to C57Bl/6N mice by intravenous injection. DNA and RNA were isolated from transduced organs for analysis by next-generation sequencing. The obtained data on DNA and RNA barcode quantities in different murine organs were analysed to assess the biodistribution and transduction efficiency of synthetic AAVs. Barcodes were identified by aligning them to the expected sequences and counted. The resulting values were normalised to the quantity of barcodes in the original library.RESULTS. Seven viral constructs based on different AAV serotypes were created as part of two AAV libraries. Six of the AAV serotypes were synthetic (sAAV1, sAAV2, sAAV3, sAAV4, sAAV5, and sAAV6). Sequencing of murine organ samples revealed significant quantities of DNA barcodes from both AAV libraries in all organs except the brain. For the L1 library, RNA barcodes were detected at a sufficient level in 4 organs, including the skeletal muscles, the heart, the liver, and the adrenal glands. For the L2 library, in addition to the listed organs, sufficient RNA-barcode levels were observed in the gonads and the kidneys. According to transduction efficiency analysis based on RNA barcode levels adjusted for DNA barcodes, sAAV5 was considered the most promising variant for gene therapy of liver-related diseases, whereas sAAV2 and sAAV6 were recognised as holding the most promise for adrenal diseases.CONCLUSIONS. The developed bioinformatics-based assessment method for synthetic AAV vector libraries can analyse AAV vector biodistribution and transduction efficiency in the body. The presented approach has the potential for selecting optimal AAV vectors for specific organs and tissues in further gene therapy development.

Characterizing Glycosylation of Adeno-Associated Virus Serotype 9 Capsid Proteins Generated from HEK293 Cells through Glycopeptide Mapping and Released Glycan Analysis.

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography-tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development.

Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

Novel chemical tyrosine functionalization of adeno-associated virus improves gene transfer efficiency in liver and retina

Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across different animal species, such as rodents, non-human primates, and humans. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy.

Hyperthermia Enhances Adeno-Associated Virus Vector Transduction Efficiency in Melanoma Cells.

Gene therapy perfectly fits in the current needs of medicine for patients with melanoma. One of the major challenges of gene therapy is to increase gene transfer. The role of hyperthermia in the improvement of AAV (adeno-associated virus) transduction efficiency has been indicated. The aim of the present study was to assess the transduction efficacy of melanoma cell lines (A375, G-361, and SK-MEL-1) with the use of the rAAV/DJ mosaic vector under hyperthermia conditions. The analysis of changes in the transduction efficacy and expression of HSPs (heat shock proteins) and receptors for AAV was performed. The transduction was performed at 37 °C and at 43 °C (1 h). Hyperthermia enhanced gene transfer in all the tested cell lines. The most efficient transducing cell line under hyperthermia was A375 (increase by 17%). G361 and SK-MEL-1 cells showed an increase of 7%. The changes in the expression of the AAV receptors and HSPs after hyperthermia were observed. A key role in the improvement of gene transfer may be played by AAVR, HSPB1, HSP6, DNAJC4, HSPD1, HSPA8, HSPA9, HSP90AB1, and AHSA1. This study showed the possibility of the use of hyperthermia as a factor enabling the stimulation of cell transduction with rAAV vectors, thereby providing tools for the improvement in the efficacy of gene therapy based on rAAV.

Cryo-EM structure and polymorphic maturation of a viral transduction enhancing amyloid fibril

Amyloid fibrils have emerged as innovative tools to enhance the transduction efficiency of retroviral vectors in gene therapy strategies. In this study, we used cryo-electron microscopy to analyze the structure of a biotechnologically engineered peptide fibril that enhances retroviral infectivity. Our findings show that the peptide undergoes a time-dependent morphological maturation into polymorphic amyloid fibril structures. The fibrils consist of mated cross-β sheets that interact by the hydrophobic residues of the amphipathic fibril-forming peptide. The now available structural data help to explain the mechanism of retroviral infectivity enhancement, provide insights into the molecular plasticity of amyloid structures and illuminate the thermodynamic basis of their morphological maturation.

Characterization of Adeno-Associated Virus Capsid Proteins by Microflow Liquid Chromatography Coupled with Mass Spectrometry.

Adeno-associated virus (AAV) has been widely used to treat various human diseases as an important delivery vector for gene therapy due to its low immunogenicity and safety. AAV capsids proteins are comprised of three capsid viral proteins (VP; VP1, VP2, VP3). The capsid proteins play a key role in viral vector infectivity and transduction efficiency. To ensure the safety and efficacy of AAV gene therapy products, the quality of AAV vector capsid proteins during development and production should be carefully monitored and controlled. Microflow liquid chromatography coupled with mass spectrometry provides superior sensitivity and fast analysis capability. It showed significant advantages in the analysis of low- concentration and large numbers of AAV samples. The intact mass of capsid protein can be accurately determined using high-resolution mass spectrometry (MS). And MS also provides highly confident confirmation of sequence coverage and post-translational modifications site identification and quantitation. In this study, we used microflow liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the characterization of AAV2 capsid protein. we obtained nearly 100% sequence coverage of low-concentration AAV2 capsid protein (8 × 1011 GC/mL). More than 30 post-translational modifications (PTMs) sites were identified, the PTMs types included deamidation, oxidation and acetylation. From this study, the proposed microflow LC-MS/MS method provides a sensitive and high throughput approach in the characterization of AAVs and other biological products with low abundance.

Points to consider for non-clinical safety evaluation of <i>in vivo</i> gene therapy products

Advances in gene therapy have led to a wide range of gene therapy products. Hence, it is challenging to use standard non-clinical safety tests, which are typically used to evaluate chemically synthesized products. Therefore, a case-by-case safety evaluation based on the unique characteristics of each product is necessary. In Japan, the fundamental considerations for evaluating the non-clinical safety of gene therapy products are outlined in “Ensuring the Quality and Safety of Gene Therapy Products”. Notably, when designing non-clinical safety studies, it is essential to consider factors such as the efficiency of vector transduction, tissue/cell tropism of the vector, and species differences in the expression level and biological activity of transgene products in the target tissue/organs. However, it is also crucial to understand the limitations of animal studies that arise due to species differences and to assess the safety of expression vectors and expressed genes based on a thorough literature review of their biological properties. The aims of this review are to present non-clinical safety evaluation approaches for gene therapy products as outlined in the Japanese guidelines and key considerations for appropriate non-clinical safety evaluation of in vivo gene therapy products.

First-in-Patient Dose Prediction for Adeno-Associated Virus-Mediated Hemophilia Gene Therapy Using Allometric Scaling.

In this study, the author compared the performance of two allometric scaling approaches and body-weight-based dose conversion approach for first-in-patient (FIP) dose prediction for adeno-associated virus (AAV)-mediated hemophilia gene therapy using preclinical and clinical efficacy data of nine AAV vectors. In general, body-weight-based direct conversion of effective doses in monkeys or dogs was more likely to underestimate FIP dose but worked for one bioengineered vector with a high transduction efficiency specifically in humans. In contrast, allometric scaling between gene efficiency factor (log GEF) and body weight (log W) was likely to overestimate FIP dose but worked for two vectors with capsid-specific T-cell responses in patients. The third approach, allometric scaling between log GEF and W-0.25 was appropriate for FIP dose prediction in the absence of T-cell responses to AAV vectors or a dramatic difference in vector transduction efficiency between animals and humans.

LBSUN178 Teniposide Improves Adeno-associated Virus-mediated Insulin Gene Therapy In Diabetic Mice.

Abstract Type 1 Diabetes Mellitus (T1DM) results from the autoimmune destruction of insulin-producing pancreatic β cells. Despite advances in our understanding of the pathogenesis and management of diabetes, as well as treatment options, which primarily include injectable insulin and whole pancreas transplant, these advancements remain unsatisfactory, with only a small percentage of patients achieving their glycemic goals. Insulin gene therapy refers to the development of β cell substitutes by introducing an insulin producing gene into endogenous cell populations that do not normally produce insulin. This strategy has the promise to overcome the limitations of current treatments. Adeno-associated viral (AAV) vector-based gene therapy holds great promise for the treatment of inherited and many complex acquired disorders. The success of AAV vectors is determined by two major factors: 1) cell transduction efficiency, and 2) persistence of the introduced gene with minimal or no host immune response following vector administration. Previous research has suggested that certain small molecules can improve AAV vector transduction efficiency and lower the vector dose below the immunological response threshold. We explore the use of teniposide, a chemotherapeutic agent, in improving the efficacy of gene therapy using AAV. We co-administered teniposide at a final dosage of 20 mg/kg with an AAV vector encoding an insulin gene controlled by a liver-specific promotor into streptozotocin (STZ) induced diabetic Hsd: ICR mice. Teniposide co-treatment produced up to a 3-fold decrease in AAV dose compared to vector alone while reducing diabetic hyperglycemia to comparable degrees as AAV delivery alone. There was no additional systemic toxicity. While the mechanism of action of teniposide-enhanced viral delivery of gene therapy is unknown, our data suggest that teniposide co-administration could significantly enhance AAV transduction in vivo and provide a novel, useful strategy to improve the clinical efficacy of gene therapy. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo

ObjectiveMelittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. MethodsThe melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. ResultsrAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. ConclusionPre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

Review on Gene Therapy on Cancer

Gene-based therapies for cancer in clinical trials include strategies that involve augmentation of immunotherapeutic and chemotherapeutic approaches. These strategies include ex vivo and in vivo cytokine gene transfer, drug sensitization with genes for prodrug delivery, and the use of drug-resistance genes for bone marrow protection from high-dose chemotherapy. Inactivation of oncogene expression and gene replacement for tumor suppressor genes are among the strategies for targeting the underlying genetic lesions in the cancer cell. A review of clinical trial results to date, primarily in patients with very advanced cancers refractory to conventional treatments, indicates that these treatments can mediate tumor regression with acceptably low toxicity. Vector development remains a critical area for future research. Important areas for future research include modifying viral vectors to reduce toxicity and immunogenicity, increasing the transduction efficiency of nonviral vectors, enhancing vector targeting and specificity, regulating gene expression, and identifying synergies between gene-based agents and other cancer therapeutics.

Polyclonality Strongly Correlates with Biological Outcomes and Is Significantly Increased Following Improvements to the Phase 1/2 HGB-206 Protocol and Manufacturing of LentiGlobin for Sickle Cell Disease (SCD; bb1111) Gene Therapy (GT)

Polyclonality Strongly Correlates with Biological Outcomes and Is Significantly Increased Following Improvements to the Phase 1/2 HGB-206 Protocol and Manufacturing of LentiGlobin for Sickle Cell Disease (SCD; bb1111) Gene Therapy (GT)

Minimal impact of ZAP on lentiviral vector production and transduction efficiency

The antiviral protein ZAP binds CpG dinucleotides in viral RNA to inhibit replication. This has likely led to the CpG suppression observed in many RNA viruses, including retroviruses. Sequences added to retroviral vector genomes, such as internal promoters, transgenes, or regulatory elements, substantially increase CpG abundance. Because these CpGs could allow retroviral vector RNA to be targeted by ZAP, we analyzed whether it restricts vector production, transduction efficiency, and transgene expression. Surprisingly, even though CpG-high HIV-1 was efficiently inhibited by ZAP in HEK293T cells, depleting ZAP did not substantially increase lentiviral vector titer using several packaging and genome plasmids. ZAP overexpression also did not inhibit lentiviral vector titer. In addition, decreasing CpG abundance in a lentiviral vector genome did not increase its titer, and a gammaretroviral vector derived from murine leukemia virus was not substantially restricted by ZAP. Overall, we show that the increased CpG abundance in retroviral vectors relative to the wild-type retroviruses they are derived from does not intrinsically sensitize them to ZAP. Further understanding of how ZAP specifically targets transcripts to inhibit their expression may allow the development of CpG sequence contexts that efficiently recruit or evade this antiviral system.

Simple RGC: ImageJ Plugins for Counting Retinal Ganglion Cells and Determining the Transduction Efficiency of Viral Vectors in Retinal Wholemounts

Simple RGC consists of a collection of ImageJ plugins to assist researchers investigating retinal ganglion cell (RGC) injury models in addition to helping assess the effectiveness of treatments. The first plugin named RGC Counter accurately calculates the total number of RGCs from retinal wholemount images. The second plugin named RGC Transduction measures the co-localisation between two channels making it possible to determine the transduction efficiencies of viral vectors and transgene expression levels. The third plugin named RGC Batch is a batch image processor to deliver fast analysis of large groups of microscope images. These ImageJ plugins make analysis of RGCs in retinal wholemounts easy, quick, consistent, and less prone to unconscious bias by the investigator. The plugins are freely available from the ImageJ update site https://sites.imagej.net/Sonjoonho/.

The Engineered MARCH8-Resistant Vesicular Stomatitis Virus Glycoprotein Enhances Lentiviral Vector Transduction.

Lentiviral vectors are one of the most commonly used viral delivery systems for gene therapy. Vesicular stomatitis virus-G envelope glycoprotein (VSV G)-pseudotyped lentiviral vectors have been widely used in clinical studies for treatment of virus infections and genetic deficient diseases. However, the efficiency of lentiviral vector transduction has been long recognized as a limiting factor in clinical gene therapy application, especially in transducing hematopoietic stem cells. MARCH8 (membrane-associated RING-CH 8), an E3 ubiquitin ligase, has been reported to target and downregulate VSV G. Results in this study show that MARCH8 induces ubiquitination and lysosome degradation of VSV G, and knockout of MARCH8 in virus-producing cells increases lentiviral vector transduction by elevating the level of VSV G protein. We then engineered VSV G mutant that has the lysine residues in the cytoplasmic domain substituted for arginine, and showed that this G mutant resists degradation by MARCH8, and allows the enhancement of transduction efficiency of lentiviral vector particles than the parental VSV G protein. This engineered VSV G mutant thus further advances the lentiviral vector system as a powerful tool in gene therapy.

Recombinant adeno-associated virus-based gene therapy combined with tissue engineering for musculoskeletal regenerative medicine.

ABSTRACTRecombinant adeno-associated viral (rAAV) vector-mediated gene delivery is a novel molecular therapeutic approach for musculoskeletal disorders which achieves tissue regeneration by delivering a transgene to the impaired tissue. In recent years, substantial scientific progress in rAAV gene therapy has led to several clinical trials for human musculoskeletal diseases. Nevertheless, there are still limitations in developing an optimal gene therapy model due to the low transduction efficiency and fast degradation of the gene vectors. To overcome the challenges of rAAV gene therapy, tissue engineering combined with gene therapy has emerged as a more promising alternative. An rAAV viral vector incorporated into a biomaterial has a more controlled gene expression, lower immune response, and higher efficiency. A number of biomaterials and architectures have been combined with rAAV viral vectors, each having its own advantages and limitations. This review aims to give a broad introduction to combinatorial therapy and the recent progress this new technology has offered.

A Novel Branched DNA-Based Flowcytometric Method for Single-Cell Characterization of Gene Therapy Products and Expression of Therapeutic Genes.

Many preclinical and clinical studies of hematopoietic stem cell-based gene therapy (GT) are based on the use of lentiviruses as the vector of choice. Assessment of the vector titer and transduction efficiency of the cell product is critical for these studies. Efficacy and safety of the modified cell product are commonly determined by assessing the vector copy number (VCN) using qPCR. However, this optimized and well-established method in the GT field is based on bulk population averages, which can lead to misinterpretation of the actual VCN per transduced cell. Therefore, we introduce here a single cell-based method that allows to unmask cellular heterogeneity in the GT product, even when antibodies are not available. We use Invitrogen’s flow cytometry-based PrimeFlow™ RNA Assay with customized probes to determine transduction efficiency of transgenes of interest, promoter strength, and the cellular heterogeneity of murine and human stem cells. The assay has good specificity and sensitivity to detect the transgenes, as shown by the high correlations between PrimeFlow™-positive cells and the VCN. Differences in promoter strengths can readily be detected by differences in percentages and fluorescence intensity. Hence, we show a customizable method that allows to determine the number of transduced cells and the actual VCN per transduced cell in a GT product. The assay is suitable for all therapeutic genes for which antibodies are not available or too cumbersome for routine flow cytometry. The method also allows co-staining of surface markers to analyze differential transduction efficiencies in subpopulations of target cells.

Mechanism for enhanced transduction of hematopoietic cells by recombinant adeno-associated virus serotype 6 vectors.

Hematopoietic gene delivery, such as hematopoietic stem/progenitor cells (HSPCs), is a promising treatment for both inherited and acquired diseases, such as hemophilia. Recently, a combined strategy to achieve more than 90% transduction efficiency was documented using recombinant adeno-associated virus serotype 6 (rAAV6) vectors. However, the mechanisms of enhanced vector transduction efficiency in hematopoietic cells are largely unknown. In this manuscript, we first reported that proteasome inhibitors, which are well-known to facilitate rAAV intracellular trafficking in various cell types, are not effective in hematopoietic cells. From the screening of small molecules derived from traditional Chinese medicine, we demonstrated that shikonin, a potential reactive oxygen species (ROS) generator, significantly increased the in vitro and ex vivo transgene expression mediated by rAAV6 vectors in hematopoietic cells, including human cord blood-derived CD34+HSPCs. Shikonin mainly targeted vector intracellular trafficking, instead of host cell entry or endonuclear single to double strand vector DNA transition, in a vector serotype-dependent manner. Moreover, a ROS scavenger completely prevented the capability of shikonin to enhance rAAV6 vector-mediated transgene expression. Taken together, these studies expand our understanding of rAAV6-mediated transduction in hematopoietic cells and are informative for improving rAAV6-based treatment of blood diseases.

Role of Essential Metal Ions in AAV Vector-Mediated Transduction

Metal elements are essential components of approximately half of all cellular proteins, and approximately one-third of all known enzymes thus far are metalloenzymes. Several cellular proteins and enzymes undoubtedly impact the transduction efficiency of recombinant adeno-associated virus (AAV) vectors, but the precise role of metal ions in this process has not been studied in detail. In the present studies, we systematically evaluated the effects of all 10 essential metal ions (calcium, cobalt, copper, iron, magnesium, manganese, molybdenum, potassium, sodium, and zinc) on the transduction efficiency of AAV vectors. We report herein that five essential metal ions (iron, magnesium, manganese, molybdenum, and sodium) had little to no effect, and calcium strongly inhibited the transduction efficiency of AAV2 vectors. Whereas copper and potassium increased the transduction efficiency by ∼5-fold and ∼2-fold, respectively, at low concentrations, both essential metals were strongly inhibitory at higher concentrations. Calcium also inhibited the transduction efficiency by ∼3-fold. Two metal ions (cobalt and zinc) increased the transduction efficiency up to ∼10-fold in a dose-dependent manner. The combined use of cobalt and zinc resulted in more than an additive effect on AAV2 vector transduction efficiency (∼30-fold). The transduction efficiency of AAV serotypes 1 through 6 (AAV1–AAV6) vectors was also augmented by zinc. Similarly, the transduction of both single-stranded (ss) and self-complementary (sc) AAV3 vectors was enhanced by zinc. Zinc treatment also led to a dose-dependent increase in expression of a therapeutic protein, the human clotting factor IX (hF.IX), mediated by scAAV3 vectors in a human hepatic cell line. This simple strategy of essential metal ion-mediated enhancement may be useful to lower the dose of AAV vectors for their optimal use in human gene therapy.