AAV-mediated gene therapies for glaucoma and uveitis: are we there yet?

Glaucoma and uveitis are non-vascular ocular diseases which are among the leading causes of blindness and visual loss. These conditions have distinct characteristics and mechanisms but share a multifactorial and complex nature, making their management challenging and burdensome for patients and clinicians. Furthermore, the lack of symptoms in the early stages of glaucoma and the diverse aetiology of uveitis hinder timely and accurate diagnoses, which are a cause of poor visual outcomes under both conditions. Although current treatment is effective in most cases, it is often associated with low patient adherence and adverse events, which directly impact the overall therapeutic success. Therefore, long-lasting alternatives with improved safety and efficacy are needed. Gene therapy, particularly utilising adeno-associated virus (AAV) vectors, has emerged as a promising approach to address unmet needs in these diseases. Engineered capsids with enhanced tropism and lower immunogenicity have been proposed, along with constructs designed for targeted and controlled expression. Additionally, several pathways implicated in the pathogenesis of these conditions have been targeted with single or multigene expression cassettes, gene editing and silencing approaches. This review discusses strategies employed in AAV-based gene therapies for glaucoma and non-infectious uveitis and provides an overview of current progress and future directions.

MicroRNAs in cancer metastasis: biological and therapeutic implications.

Cancer metastasis is the primary cause of cancer-related deaths. The seeding of primary tumours at a secondary site is a highly inefficient process requiring substantial alterations in the genetic architecture of cancer cells. These alterations include significant changes in global gene expression patterns. MicroRNAs are small, non-protein coding RNAs which play a central role in regulating gene expression. Here, we focus on microRNA determinants of cancer metastasis and examine microRNA dysregulation in metastatic cancer cells. We dissect the metastatic process in a step-wise manner and summarise the involvement of microRNAs at each step. We also discuss the advantages and limitations of different microRNA-based strategies that have been used to target metastasis in pre-clinical models. Finally, we highlight current clinical trials that use microRNA-based therapies to target advanced or metastatic tumours.

Clinical and economic burden of benign and malignant skin lesions in renal transplant recipients.

BACKGROUND: Studies evaluating the economic burden of dermatological care in the transplant setting are currently not available in Australia. AIMS: To evaluate the clinical and economic burden of benign and malignant skin lesions in renal transplant recipients in Central Queensland. METHODS: A bottom-up approach was used to determine the clinical burden and direct costs from patient-level Medicare data obtained from Service Australia for skin lesions. RESULTS: Seventy-six percent of the renal transplant population in Central Queensland participated in this study. The median age was 57.0 years (standard deviation ± 13.6) and the majority (61.8%) of participants were men. The mean duration after transplant surgery was 99.9 months (interquartile range, 73.2-126.6 months). During a 2-year follow-up, 22 (40%) patients were diagnosed with benign skin lesions, 21 (38%) with nonmelanoma skin carcinoma (NMSC) and one (2%) with melanoma. There was a total of 231 visits to clinicians for diagnostic and therapeutic skin procedures and the direct costs to Medicare was $48 806 Australian Dollars (AUD) or $30 427 US Dollars (USD). Approximately 86% of the total direct costs was spent for nonNMSC and mean direct costs for NMSC was $763 AUD (or $476 USD). CONCLUSION: This Medicare data-based study provides further insight into the burgeoning clinical and economic burden of the care for benign and malignant skin lesions in the renal transplantation setting in Australia.

Neutralisation of adeno-associated virus transduction by human vitreous humour.

Neutralising antibodies (NAbs), caused by past adeno-associated virus (AAV) infection, represent a critical challenge for AAV-mediated gene therapy, with even low NAb titres capable of inhibiting gene transfer, however in protein-rich environments such as the vitreous it is expected that other constituents could also interact with the transduction process. Inhibition of AAV2/2, AAV2/5, AAV2/6 and AAV2/8 transduction by human vitreous humour (VH) obtained from 80 post-mortem eye cups was investigated in this report, with clinically relevant vitreous dilutions as low as 1:2. Unexpectedly, the highest prevalence of inhibition of transduction was observed against AAV2/6, with 66% of tested samples displaying neutralisation at a 1:2 VH dilution. Only two samples showed inhibition of AAV2/8, indicating this serotype is an attractive vector for use in non-vitrectomised eyes of unscreened individuals. Levels of anti-AAV NAbs observed in the VH were much lower than previously observed in serum of a similar Australian population. Among ten tested eye cup pairs, we observed only small variation in anti-AAV NAbs levels between the left and right eye cups. Interaction with 1:2 diluted VH had an augmentation effect on AAV2/8 transduction (p = 0.004), a phenomenon which was not due to albumin or transferrin and which, if developed, might benefit the use of AAV2/8 in clinical settings.

Stathmin 1 deficiency induces erythro-megakaryocytic defects leading to macrocytic anemia and thrombocythemia in Stathmin 1 knock out mice.

Stathmin 1 (STMN1) is a cytosolic phosphoprotein that was discovered as a result of its high level of expression in leukemic cells. It plays an important role in the regulation of mitosis by promoting depolymerization of the microtubules that make up the mitotic spindle and, aging has been shown to impair STMN1 levels and change microtubule stability. We have previously demonstrated that a high level of STMN1 expression during early megakaryopoiesis is necessary for proliferation of megakaryocyte progenitors and that down-regulation of STMN1 expression during late megakaryopoiesis is important for megakaryocyte maturation and platelet production. In this report, we examined the effects of STMN1 deficiency on erythroid and megakaryocytic lineages in the mouse. Our studies show that STMN1 deficiency results in mild thrombocytopenia in young animals which converts into profound thrombocythemia as the mice age. STMN1 deficiency also lead to macrocytic changes in both erythrocytes and megakaryocytes that persisted throughout the life of STMN1 knock-out mice. Furthermore, STMN1 knock-out mice displayed a lower number of erythroid and megakaryocytic progenitor cells and had delayed recovery of their blood counts after chemotherapy. These studies show an important role for STMN1 in normal erythro-megakaryopoietic development and suggests potential implications for disorders affecting these hematopoietic lineages.

Expression system for structural and functional studies of human glycosylation enzymes.

Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

Adeno-associated virus neutralising antibodies in type 1 diabetes mellitus.

Recombinant Adeno-associated viruses (AAVs) are an attractive vector for gene therapy delivery which may be blocked by AAV neutralising antibodies (NAbs). As Type 1 Diabetes (T1DM) is an endocrine disease of immunological origin, it is likely that NAb profiles are altered in the disease. In this study NAb to AAV2, AAV5, AAV6, and AAV8 in 72 subjects with T1DM and 45 non-diabetic patients were measured over a 4-year follow-up period. AAV2 NAb titres were significantly lower in non-diabetic subjects (P = 0.036). The T1DM group had more AAV8 NAb activity at baseline (P = 0.019), whilst after 4 years follow-up the T1DM group displayed developed increased AAV 5 (P = 0.03), 6 (P = 0.03) and 8 (P = 0.002) activity relative to the control group, however, overall AAV5 and 8 NAb levels were very low in patients <40. AAV NAb titre activity and prevalence generally appears higher in T1DM, however, low levels of AAV 5 and 8, particular in younger adult age groups at which T1DM can be targeted, could make these attractive vectors to target the disease.

Identification of Antibody Targets for Tuberculosis Serology using High-Density Nucleic Acid Programmable Protein Arrays.

Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n = 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n = 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n = 244) and heretofore untested samples (n = 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients' TB status according to geography (US or SA) and HIV (HIV- or HIV+) status. With ROC curve analysis under cross validation, the classifiers performed with an AUC for US/HIV- at 0.807; US/HIV+ at 0.782; SA/HIV- at 0.868; and SA/HIV+ at 0.723. With this study we demonstrate a new platform for biomarker/antibody screening and delineate its utility to identify previously unknown immunoreactive proteins.

Retinal explant culture: A platform to investigate human neuro-retina.

The retina is the tissue responsible for light detection, in which retinal neurons convert light energy into electrical signals to be transported towards the visual cortex. Damage of retinal neurons leads to neuronal cell death and retinal pathologies, compromising visual acuity and eventually leading to irreversible blindness. Models of retinal neurodegeneration include 2D systems like cell lines, disassociated cultures and co-cultures, and 3D models like organoids, organotypic retinal cultures and animal models. Of these, ex vivo human retinal cultures are arguably the most suitable models for translational research as they retain complex inter-cellular interactions of the retina and precisely mimic in-situ responses. In this review, we summarize the distinguishing features of the human retina which are important to preserve in experimental culture, the historical development of human retinal culture systems, the factors affecting ex vivo human retinal culture and the applications and challenges associated with current methods of human retinal explant culture.

Gene therapy and the adeno-associated virus in the treatment of genetic and acquired ophthalmic diseases in humans: Trials, future directions and safety considerations.

Voretigene neparvovec-rzyl was recently approved for the treatment of Leber congenital amaurosis, and the use of gene therapy for eye disease is attracting even greater interest. The eye has immune privileged status, is easily accessible, requires a reduced dosage of therapy due to its size and is highly compartmentalized, significantly reducing systemic spread. Adeno-associated virus (AAV), with its low pathogenicity, prolonged expression profile and ability to transduce multiple cell types, has become the leading gene therapy vector. Target diseases have moved beyond currently untreatable inherited dystrophies to common, partially treatable acquired conditions such as exudative age-related macular degeneration and glaucoma, but use of the technology in these conditions imposes added obligations for caution in vector design. This review discusses the current status of AAV gene therapy trials in genetic and acquired ocular diseases, and explores new scientific developments, which could help ensure effective and safe use of the therapy in the future.

Evaluation of the Glypican 3 promoter for transcriptional targeting of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a major health problem as evidenced by its increasing incidence and high morbidity and mortality rates. Most patients with HCC have underlying liver disease and dysfunction which limits the current therapeutic options. Treatments that spare the liver and destroy the HCC are needed. Targeting transcriptional differences between HCC and liver cells may provide this therapeutic window. In this study, we examine the potential of the Glypican 3 (GPC3) promoter as a targeting strategy. GPC3 is an oncofetal protein belonging to the proteoglycan family which is normally only expressed during fetal development. However, in HCC, the expression of this protein is reactivated. Here, we show that GPC3 is expressed primarily in HCC and not in normal liver lines. We show that the GPC3 promoter can be used to drive expression of significantly more luciferase and eYFP in HCC cell lines compared to normal liver cells. Further, we show that vectors containing cytosine deaminase (CD) under GPC3 promotor control induced significantly more killing of HCC cell lines after treatment with 5-FC compared to normal liver cell lines. These data suggest that transcriptionally targeted delivery of transgene in HCC cells can be achieved using the GPC3 promoter and this targeting strategy produces limited toxicity to normal liver cells.

Response of skeletal muscle UCP2-expression during metabolic adaptation to caloric restriction.

BACKGROUND/OBJECTIVES: Spendthrift vs. thrifty individuals expend more energy and experience greater weight loss during caloric restriction (CR). Adaptive mechanisms in skeletal muscle, adipose tissue, and on hormone level modulate energy expenditure (EE) during weight loss. Metabolic mechanisms underlying the variability in EE during CR are unclear. The present study explored whether during long-term CR (i) gene expression changes in skeletal muscle and adipose tissue relate with the individual EE response and weight loss, and (ii) altered catecholamine and FGF21-concentrations are associated with measures of metabolic adaptation. SUBJECTS/METHODS: In a 10-week inpatient study, 24-h EE was measured before and after 6 weeks of 50% CR in 12 subjects using whole-room indirect calorimetry. Weight loss was assessed and repeated hormone measurements performed. Muscle and adipose tissue biopsies were taken before and after CR, and gene expression was assessed (RNA-Seq). Genes showing the most significant changes after CR were tested for association with EE and followed-up for further association with metabolic measures in a separate phenotyping study (n = 103). RESULTS: Muscle UCP2 showed the strongest change after CR (log2-fold change = -1.57, false discovery rate = 0.10) and was considered the best gene for exploration of metabolic adaptive processes. A greater decrease in UCP2-expression was associated with less weight loss (P = 0.03, r = 0.77) and relatively lower 24-h EE after CR (P = 0.001, r = -0.96). Post-CR changes in FGF21-plasma concentrations correlated with UCP2-expression change (P = 0.02, r = -0.89) and weight loss (P = 0.003, r = -0.83). In a separate metabolic phenotyping study, muscle UCP2-expression correlated with respiratory quotient and macronutrient oxidation. In adipose tissue, no candidate genes for metabolic exploration were found. CONCLUSIONS: Changes in muscle UCP2-expression reflect an inter-individual metabolic response to long-term CR and may influence EE and weight loss via modulation of substrate oxidation.

Reduction of MHC-I expression limits T-lymphocyte-mediated killing of Cancer-initiating cells.

BACKGROUND: It has been proposed that cancer establishment, maintenance, and recurrence may be attributed to a unique population of tumor cells termed cancer-initiating cells (CICs) that may include characteristics of putative cancer stem cell-like cells. Studies in lung cancer have shown that such cells can be enriched and propagated in vitro by culturing tumor cells in serum-free suspension as tumorspheres. CICs have been characterized for their phenotype, stem cell-like qualities, and their role in establishing tumor and maintaining tumor growth. Less is known about the interaction of CICs with the immune system. METHODS: We established CIC-enriched tumorspheres from murine TC-1 lung cancer cells, expressing human papillomavirus 16 (HPV-16) E6/E7 antigens, and evaluated their susceptibility to antitumor immune responses both in vitro and in vivo. RESULTS: TC-1 CICs demonstrated reduced expression of surface major histocompatibility complex (MHC)-I molecules compared to non-CICs. We similarly determined decreased MHC-I expression in five of six human lung cancer cell lines cultured under conditions enriching for CICs. In vivo, TC-1 cells enriched for CICs were resistant to human papillomavirus 16 E6/E7 peptide vaccine-mediated killing. We found that vaccinated mice challenged with CIC enriched tumorspheres demonstrated shorter survivals and showed significantly fewer CD8+ tumor infiltrating lymphocytes compared to CIC non-enriched challenged mice. Furthermore, cultured cytotoxic T lymphocytes (CTLs) from vaccinated mice demonstrated reduced capacity to lyse TC-1 cells enriched for CICs compared to non-enriched TC-1 cells. Following treatment with IFN-γ, both CIC enriched and non-enriched TC-1 cells expressed similar levels of MHC-I, and the increased MHC-I expression on CICs resulted in greater CTL-mediated tumor lysis and improved tumor-free survival in mice. CONCLUSIONS: These results suggest that the attenuated expression of MHC-I molecules by CICs represents a potential strategy of CICs to escape immune recognition, and that the development of successful immunotherapy strategies targeting CICs may decrease their resistance to T cell-mediated immune detection by enhancing CIC MHC-I expression.

MicroRNA-Regulated Gene Delivery Systems for Research and Therapeutic Purposes.

Targeted gene delivery relies on the ability to limit the expression of a transgene within a defined cell/tissue population. MicroRNAs represent a class of highly powerful and effective regulators of gene expression that act by binding to a specific sequence present in the corresponding messenger RNA. Involved in almost every aspect of cellular function, many miRNAs have been discovered with expression patterns specific to developmental stage, lineage, cell-type, or disease stage. Exploiting the binding sites of these miRNAs allows for construction of targeted gene delivery platforms with a diverse range of applications. Here, we summarize studies that have utilized miRNA-regulated systems to achieve targeted gene delivery for both research and therapeutic purposes. Additionally, we identify criteria that are important for the effectiveness of a particular miRNA for such applications and we also discuss factors that have to be taken into consideration when designing miRNA-regulated expression cassettes.

DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research.

The mission of the DNASU Plasmid Repository is to accelerate research by providing high-quality, annotated plasmid samples and online plasmid resources to the research community through the curated DNASU database, website and repository (http://dnasu.asu.edu or http://dnasu.org). The collection includes plasmids from grant-funded, high-throughput cloning projects performed in our laboratory, plasmids from external researchers, and large collections from consortia such as the ORFeome Collaboration and the NIGMS-funded Protein Structure Initiative: Biology (PSI:Biology). Through DNASU, researchers can search for and access detailed information about each plasmid such as the full length gene insert sequence, vector information, associated publications, and links to external resources that provide additional protein annotations and experimental protocols. Plasmids can be requested directly through the DNASU website. DNASU and the PSI:Biology-Materials Repositories were previously described in the 2010 NAR Database Issue (Cormier, C.Y., Mohr, S.E., Zuo, D., Hu, Y., Rolfs, A., Kramer, J., Taycher, E., Kelley, F., Fiacco, M., Turnbull, G. et al. (2010) Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res., 38, D743-D749.). In this update we will describe the plasmid collection and highlight the new features in the website redesign, including new browse/search options, plasmid annotations and a dynamic vector mapping feature that was developed in collaboration with LabGenius. Overall, these plasmid resources continue to enable research with the goal of elucidating the role of proteins in both normal biological processes and disease.

Simultaneous inhibition of two regulatory T-cell subsets enhanced Interleukin-15 efficacy in a prostate tumor model.

IL-15 has potential as an immunotherapeutic agent for cancer treatment because of its ability to effectively stimulate CD8 T cell, natural killer T cell, and natural killer cell immunity. However, its effectiveness may be limited by negative immunological checkpoints that attenuate immune responses. Recently a clinical trial of IL-15 in cancer immunotherapy was initiated. Finding strategies to conquer negative regulators and enhance efficacy of IL-15 is critical and meaningful for such clinical trials. In a preclinical study, we evaluated IL-15 combined with antibodies to block negative immune regulator cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) in an established murine transgenic adenocarcinoma of mouse prostate (TRAMP)-C2 prostate tumor model. IL-15 treatment resulted in a significant prolongation of survival in tumor-bearing animals. Coadministration of anti-PD-L1 or anti-CTLA-4 singly with IL-15 did not improve animal survival over that of IL-15 alone. However, simultaneous administration of IL-15 with anti-CTLA-4 and anti-PD-L1 was associated with increased numbers of tumor antigen-specific tetramer-positive CD8 T cells, increased CD8 T-cell tumor lytic activity, augmented antigen-specific IFN-γ release, decreased rates of tumor growth, and improved animal survival compared with IL-15 alone. Furthermore, triple combination therapy was associated with inhibition of suppressive functions of CD4(+)CD25(+) regulatory T cells and CD8(+)CD122(+) regulatory T cells. Thus, simultaneous blockade of CTLA-4 and PD-L1 protected CD4 and/or CD8 T-cell activity from these regulatory T cells. Combining the immune stimulatory properties of IL-15 with simultaneous removal of two critical immune inhibitory checkpoints, we showed enhancement of immune responses, leading to increased antitumor activity.

Oral vaccination with adeno-associated virus vectors expressing the Neu oncogene inhibits the growth of murine breast cancer.

Recombinant adeno-associated viruses (AAV) have been used for therapeutic gene transfer. These vectors offer a number of advantages including resistance to the effects of pH, a broad cellular tropism, efficient gene transfer, persistence of gene expression, and little toxicity. AAV vectors; however, at high doses can induce humoral and cellular immune responses. While potentially problematic for replacement gene therapy, this effect may be advantageous for antitumor vaccination. We examined the activity of an oral and intramuscular antitumor vaccination using AAV serotypes 5 and 6 expressing a truncated neu oncogene in a neu-positive murine TUBO breast cancer model. Mice receiving a single oral administration of AAV5-neu or AAV6-neu demonstrated improved survival. Oral vaccination significantly improved survivals compared with intramuscular vaccination. Mice vaccinated with AAV6-neu survived longer than those treated with AAV5-neu. Vaccination with AAV5-neu or AAV6-neu induced both humoral and cellular immune responses against the NEU antigen. These responses were more robust in the mice undergoing oral vaccination compared with mice receiving the intramuscular vaccination. Protection from tumor was long lasting with 80% of the animals treated with oral AAV6-neu surviving a re-challenge with TUBO cells at 120 and 320 days post-vaccination. Further evaluation of AAV-based vectors as tumor vaccines is warranted.

AAV-Based Strategies for Treatment of Retinal and Choroidal Vascular Diseases: Advances in Age-Related Macular Degeneration and Diabetic Retinopathy Therapies.

Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are vascular diseases with high prevalence, ranking among the leading causes of blindness and vision loss worldwide. Despite being effective, current treatments for AMD and DR are burdensome for patients and clinicians, resulting in suboptimal compliance and real risk of vision loss. Thus, there is an unmet need for long-lasting alternatives with improved safety and efficacy. Adeno-associated virus (AAV) is the leading vector for ocular gene delivery, given its ability to enable long-term expression while eliciting relatively mild immune responses. Progress has been made in AAV-based gene therapies for not only inherited retinal diseases but also acquired conditions with preclinical and clinical studies of AMD and DR showing promising results. These studies have explored several pathways involved in the disease pathogenesis, as well as different strategies to optimise gene delivery. These include engineered capsids with enhanced tropism to particular cell types, and expression cassettes incorporating elements for a targeted and controlled expression. Multiple-acting constructs have also been investigated, in addition to gene silencing and editing. Here, we provide an overview of strategies employing AAV-mediated gene delivery to treat AMD and DR. We discuss preclinical efficacy studies and present the latest data from clinical trials for both diseases.

Incremental increases in physiological fluid shear progressively alter pathogenic phenotypes and gene expression in multidrug resistant Salmonella.

The ability of bacteria to sense and respond to mechanical forces has important implications for pathogens during infection, as they experience wide fluid shear fluctuations in the host. However, little is known about how mechanical forces encountered in the infected host drive microbial pathogenesis. Herein, we combined mathematical modeling with hydrodynamic bacterial culture to profile transcriptomic and pathogenesis-related phenotypes of multidrug resistant S. Typhimurium (ST313 D23580) under different fluid shear conditions relevant to its transition from the intestinal tract to the bloodstream. We report that D23580 exhibited incremental changes in transcriptomic profiles that correlated with its pathogenic phenotypes in response to these progressive increases in fluid shear. This is the first demonstration that incremental changes in fluid shear forces alter stress responses and gene expression in any ST313 strain and offers mechanistic insight into how forces encountered by bacteria during infection might impact their disease-causing ability in unexpected ways.

High-throughput cloning and expression library creation for functional proteomics.

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single-gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator(TM) DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12).