• Cell therapy
• Gene therapy
• NAFLD
• Non-Alcoholic Fatty Liver Disease
• Stem cell
• Viral vectors

• Humans
• Non-alcoholic Fatty Liver Disease/therapy
• Non-alcoholic Fatty Liver Disease/genetics
• Non-alcoholic Fatty Liver Disease/immunology
• Genetic Therapy/methods
• Hepatocytes/metabolism
• Hepatocytes/cytology
• Animals
• Cell- and Tissue-Based Therapy/methods
• Endoplasmic Reticulum Stress
• Hepatic Stellate Cells/metabolism
• Liver/pathology

• CP: Stem cell research
• in vivo gene therapy
• large animal models
• lentiviral vectors
• liver damage
• liver organoids
• liver stem cells
• translational research

• Animals
• Hepatocytes/metabolism
• Lentivirus/genetics
• Liver/metabolism
• Mice
• Gene Transfer Techniques
• Homeostasis
• Epithelial Cells/metabolism
• Mice, Inbred C57BL
• Bile Ducts/metabolism
• Organoids/metabolism
• Genetic Vectors
• Male

• AAV
• CRISPR
• LM-PCR
• S-EPTS
• double-strand DNA break
• factor IX
• hemophilia
• off-target
• whole genome sequencing

• gene augmentation
• genome editing
• inherited liver diseases

• Humans
• Genetic Therapy/methods
• Gene Editing/methods
• Liver Diseases/therapy
• Liver Diseases/genetics
• Animals
• Genetic Vectors/genetics

• Envelope Engineering
• Hemophilia A
• In Vivo Gene Therapy
• Lentiviral Vectors
• Liver Endothelial Cells

• Animals
• Hemophilia A/therapy
• Hemophilia A/genetics
• Genetic Vectors/genetics
• Endothelial Cells/metabolism
• Mice
• Lentivirus/genetics
• Genetic Therapy/methods
• Liver/metabolism
• Factor VIII/genetics
• Factor VIII/metabolism
• Humans
• Viral Envelope Proteins/genetics
• Viral Envelope Proteins/metabolism
• Transduction, Genetic/methods
• Hepatocytes/metabolism
• Hepatocytes/virology
• Membrane Glycoproteins/genetics
• Membrane Glycoproteins/metabolism

• SR-Tiget Core Grant TTACC0422TT Fondazione Telethon (FT)
• UPGRADE European Commission (EC)

• AAV
• CRISPR
• factor IX
• factor VIII
• gene editing
• hemophilia

• United States
• Adult
• Child
• Humans
• Hemophilia A/genetics
• Hemophilia A/therapy
• Hemophilia B/genetics
• Hemophilia B/therapy
• DNA, Complementary
• Genetic Therapy

• DNA translocation
• HiFi-Cas9
• Target-seq
• off-target
• viral integration

• DOPA decarboxylase
• Parkinson’s disease
• chemogenetics
• promoter

• Rats
• Animals
• Dopaminergic Neurons/metabolism
• Dopamine/metabolism
• Dopa Decarboxylase/metabolism
• Pars Compacta/metabolism
• Parkinsonian Disorders/metabolism
• Parkinson Disease/metabolism
• Tyrosine 3-Monooxygenase/genetics
• Tyrosine 3-Monooxygenase/metabolism
• Substantia Nigra/metabolism

• CRISPR-Cas9
• DNA damage response
• DNA double-strand breaks
• adeno-associated vector
• gene editing
• genotoxicity
• hematopoietic stem/progenitor cells
• integrase-defective lentiviral vector
• inverted terminal repeats
• p53 pathway

• CRISPR-Cas9
• Sleeping Beauty
• T cell receptor
• chimeric antigen receptor
• leukemia
• transposon

• Animals
• Antigens, CD19
• CRISPR-Cas Systems
• Graft vs Host Disease/metabolism
• Hematopoietic Stem Cell Transplantation
• Immunotherapy
• Immunotherapy, Adoptive
• Mice
• Neoplasms/genetics
• Neoplasms/therapy
• Receptors, Antigen, T-Cell/metabolism
• Receptors, Chimeric Antigen/genetics
• Receptors, Chimeric Antigen/metabolism
• T-Lymphocytes

• clinical trials
• disease diagnosis
• disease modeling
• gene therapy
• genome editing
• immunotherapy

• National Natural Science Foundation of China
• National Key Research and Development Program of China
• Key Research and Development Program of Sichuan Province

There are many clinical trials underway for the development of gene therapies, and some have resulted in gene therapy products being commercially approved already. Significant progress was made to develop safer and more effective strategies to deliver and regulate genetic products. An unsolved aspect is the immune system, which can affect the efficiency of gene therapy in different ways. Here we present an overview of approved gene therapy products and the immune response elicited by gene delivery systems. These include responses against the vector or its content after delivery and against the product of the corrected gene. Strategies to overcome the hurdles include hiding the vector or/and the transgene product from the immune system and hiding the immune system from the vector/transgene product. Combining different strategies, such as patient screening and intelligent vector design, gene therapy is set to make a difference in the life of patients with severe genetic diseases.

Insulin is the primary autoantigen (Ag) targeted by T cells in type 1 diabetes (T1D). Although biomarkers precisely identifying subjects at high risk of T1D are available, successful prophylaxis is still an unmet need. Leaky central tolerance to insulin may be partially ascribed to the instability of the MHC-InsB_9-23 complex, which lowers TCR avidity, thus resulting in defective negative selection of autoreactive clones and inadequate insulin-specific T regulatory cell (Treg) induction. We developed a lentiviral vector (LV)-based strategy to engineer thymic epithelial cells (TECs) to correct diabetogenic T cell repertoire. Intrathymic (it) LV injection established stable transgene expression in EpCAM⁺ TECs, by virtue of transduction of TEC precursors. it-LV-driven presentation of the immunodominant portion of ovalbumin allowed persistent and complete negative selection of responsive T cells in OT-II chimeric mice. We successfully applied this strategy to correct the diabetogenic repertoire of young non-obese diabetic mice, imposing the presentation by TECs of the stronger agonist InsulinB_9-23R22E and partially depleting the existing T cell compartment. We further circumscribed LV-driven presentation of InsulinB_9-23R22E by micro-RNA regulation to CD45⁻ TECs without loss of efficacy in protection from diabetes, associated with expanded insulin-specific Tregs. Overall, our gene transfer-based prophylaxis fine-tuned the central tolerance processes of negative selection and Treg induction, correcting an autoimmune prone T cell repertoire.

• T regulatory cells
• autoimmunity
• central tolerance
• in vivo gene transfer
• lentiviral vector
• negative selection
• preventive immunotherapy
• thymus
• type 1 diabetes

Lentiviral vectors (LVs) play an important role in gene therapy and have proven successful in clinical trials. LVs are capable of integrating specific genetic materials into the target cells and allow for long-term expression of the cDNA of interest. The use of non-integrating LVs (NILVs) reduces insertional mutagenesis and the risk of malignant cell transformation over integrating lentiviral vectors. NILVs enable transient expression or sustained episomal expression, especially in non-dividing cells. Important modifications have been made to the basic human immunodeficiency virus (HIV) structures to improve the safety and efficacy of LVs. NILV-aided transient expression has led to more pre-clinical studies on primary immunodeficiencies, cytotoxic cancer therapies, and hemoglobinopathies. Recently, the third generation of self-inactivating LVs was applied in clinical trials for recombinant protein production, vaccines, gene therapy, cell imaging, and induced pluripotent stem cell (iPSC) generation. This review discusses the basic lentiviral biology and the four systems used for generating NILV designs. Mutations or modifications in LVs and their safety are addressed with reference to pre-clinical studies. The detailed application of NILVs in promising pre-clinical studies is also discussed.

• Animals
• Autoimmunity/drug effects
• Diabetes Mellitus, Type 1/immunology
• Diabetes Mellitus, Type 1/prevention & control
• Diabetes Mellitus, Type 1/surgery
• Gene Transfer Techniques
• Graft Survival/immunology
• Hepatocytes/immunology
• Hepatocytes/metabolism
• Insulin-Secreting Cells/immunology
• Insulin-Secreting Cells/metabolism
• Islets of Langerhans Transplantation/immunology
• Islets of Langerhans Transplantation/methods
• Mice
• Mice, Inbred NOD
• Peptides/therapeutic use
• Recurrence
• Secondary Prevention
• T-Lymphocytes, Regulatory/immunology

Delivering rapid protection against infectious agents to non-immune populations is a formidable public health challenge. Although passive immunotherapy is a fast and effective method of protection, large-scale production and administration of monoclonal antibodies (mAbs) is expensive and unpractical. Viral vector-mediated delivery of mAbs offers an attractive alternative to their direct injection. Integrase-defective lentiviral vectors (IDLV) are advantageous for this purpose due to the absence of pre-existing anti-vector immunity and the safety features of non-integration and non-replication. We engineered IDLV to produce the humanized mAb VN04-2 (IDLV-VN04-2), which is broadly neutralizing against H5 influenza A virus (IAV), and tested the vectors’ ability to produce antibodies and protect from IAV in vivo. We found that IDLV-transduced cells produced functional VN04-2 mAbs in a time- and dose-dependent fashion. These mAbs specifically bind the hemagglutinin (HA), but not the nucleoprotein (NP) of IAV. VN04-2 mAbs were detected in the serum of mice at different times after intranasal (i.n.) or intramuscular (i.m.) administration of IDLV-VN04-2. Administration of IDLV-VN04-2 by the i.n. route provided rapid protection against lethal IAV challenge, although the protection did not persist at later time points. Our data suggest that administration of mAb-expressing IDLV may represent an effective strategy for rapid protection against infectious diseases.

• genetic immunization
• influenza
• integrase-defective lentiviral vector
• monoclonal antibody
• passive immunity

• CRISPR/Cas9
• TALENs
• genome editing
• liver-targeted gene therapy
• viral vectors
• zinc finger nucleases

• Animals
• Biomarkers
• Brain Diseases, Metabolic/genetics
• Brain Diseases, Metabolic/therapy
• CRISPR-Cas Systems
• Clinical Studies as Topic
• Disease Management
• Drug Evaluation, Preclinical
• Gene Editing
• Gene Expression
• Gene Transfer Techniques
• Genetic Predisposition to Disease
• Genetic Therapy/methods
• Genetic Vectors/classification
• Genetic Vectors/genetics
• Humans
• Liver Diseases/genetics
• Liver Diseases/therapy
• Organ Specificity/genetics
• Transduction, Genetic
• Treatment Outcome

• azacytidine
• gene expression
• lentiviral transduction
• liver progenitor cell
• liver-enriched transcription factor
• microRNA
• rat liver epithelial cells

• Animals
• Animals, Newborn
• Azacitidine/pharmacology
• Cell Differentiation/drug effects
• Cell Proliferation/drug effects
• Epigenesis, Genetic/genetics
• Epithelial Cells/drug effects
• Gene Expression Profiling/methods
• Gene Expression Regulation/drug effects
• Hepatocyte Nuclear Factor 4/genetics
• Liver/drug effects
• Liver/metabolism
• MicroRNAs/genetics
• RNA, Messenger/genetics
• Rats
• Stem Cells/drug effects
• Transduction, Genetic

Several viral vector-based gene therapy drugs have now received marketing approval. A much larger number of additional viral vectors are in various stages of clinical trials for the treatment of genetic and acquired diseases, with many more in pre-clinical testing. Efficiency of gene transfer and ability to provide long-term therapy make these vector systems very attractive. In fact, viral vector gene therapy has been able to treat or even cure diseases for which there had been no or only suboptimal treatments. However, innate and adaptive immune responses to these vectors and their transgene products constitute substantial hurdles to clinical development and wider use in patients. This review provides an overview of the type of immune responses that have been documented in animal models and in humans who received gene transfer with one of three widely tested vector systems, namely adenoviral, lentiviral, or adeno-associated viral vectors. Particular emphasis is given to mechanisms leading to immune responses, efforts to reduce vector immunogenicity, and potential solutions to the problems. At the same time, we point out gaps in our knowledge that should to be filled and problems that need to be addressed going forward.

• adaptive immunity
• adeno-associated virus
• adenovirus
• innate immunity
• lentivirus

Long noncoding RNAs (lncRNAs) are emerging as important regulators of numerous biological processes, especially in cancer development. Aberrantly expressed and specifically located in tumor cells, they exert distinct functions in different cancers via regulating multiple downstream targets such as chromatins, RNAs, and proteins. Differentiation antagonizing non-protein coding RNA (DANCR) is a cytoplasmic lncRNA that generally works as a tumor promoter. Mechanically, DANCR promotes the functions of vital components in the oncogene network by sponging their corresponding microRNAs or by interacting with various regulating proteins. DANCR's distinct expression in tumor cells and collective involvement in pro-tumor pathways make it a promising therapeutic target for broad cancer treatment. Herein, we summarize the functions and molecular mechanism of DANCR in human cancers. Furthermore, we introduce the use of CRISPR/Cas9, antisense oligonucleotides and small interfering RNAs as well as viral, lipid, or exosomal vectors for onco-lncRNA targeted treatment. Conclusively, DANCR is a considerable promoter of cancers with a bright prospect in targeted therapy.

• DANCR
• cancer
• long non-coding RNA
• mechanism
• therapy

The adaptation of CRISPR/Cas technology for use in mammals has revolutionized genome engineering. In particular with regard to clinical application, efficient expression of Cas9 within a narrow time frame is highly desirable to minimize the accumulation of off-target editing. We developed an effective, aptamer-independent retroviral delivery system for Cas9 mRNAs that takes advantage of a unique foamy virus (FV) capability: the efficient encapsidation and transfer of non-viral RNAs. This enabled us to create a FV vector toolbox for efficient, transient delivery (TraFo) of CRISPR/Cas9 components into different target tissues. Co-delivery of Cas9 mRNA by TraFo-Cas9 vectors in combination with retroviral, integration-deficient single guide RNA (sgRNA) expression enhanced efficacy and specificity of gene-inactivation compared with CRISPR/Cas9 lentiviral vector systems. Furthermore, separate TraFo-Cas9 delivery allowed the optional inclusion of a repair matrix for efficient gene correction or tagging as well as the addition of fluorescent negative selection markers for easy identification of off-target editing or incorrect repair events. Thus, the TraFo CRISPR toolbox represents an interesting alternative technology for gene inactivation and gene editing.

• CAR T-cell
• CRISPR/Cas9
• TraFo-Cas9
• foamy virus
• genome engineering
• transient system
• viral vector

Gene expression regulation is the result of complex interactions between transcriptional and post-transcriptional controls, resulting in cell-type-specific gene expression patterns that are determined by the developmental and differentiation stage of pathophysiological conditions. Understanding the complexity of gene expression regulatory networks is fundamental to gene therapy, an approach which has the potential to treat and cure inherited disorders by delivering the correct gene to patient specific cells or tissues by means of both viral and non-viral vectors. Besides the issues of biosafety, in recent years efforts have focused on achieving a robust and sustained transgene expression, which attains a phenotypic correction in several diseases, while avoiding transgene-related adverse effects, such as overexpression-associated cytotoxicity and/or immune responses to the transgene. In this sense, the use of cell-type-specific promoters and microRNA target sequences (miRTs) in gene transfer expression cassettes have allowed for a restricted expression after gene transfer in several studies. This review will focus on the use of transcriptional and post-transcriptional regulation to achieve a highly specific and safe transgene expression, as well as their application in ex vivo and in vivo gene therapeutic approaches.

• IFITM3
• cyclosporine
• gene editing
• gene therapy
• hematopoietic stem cells
• innate immunity
• lentiviral vectors
• transduction enhancers

• CRISPR-Cas Systems
• Cells, Cultured
• Child
• Female
• Gene Editing/methods
• Humans
• Induced Pluripotent Stem Cells/metabolism
• Middle Aged
• Muscle Development/genetics
• Myoblasts/metabolism
• Myotonic Dystrophy/genetics
• Myotonic Dystrophy/metabolism
• Myotonic Dystrophy/pathology
• Trinucleotide Repeat Expansion/genetics

• DP1 GM105378 NIGMS NIH HHS

• aortic aneurysm
• disease models
• gene therapy
• lentiviral vectors
• viral targeting

Immunotherapy is emerging as a new pillar of cancer treatment with potential to cure. However, many patients still fail to respond to these therapies. Among the underlying factors, an immunosuppressive tumor microenvironment (TME) plays a major role. Here we show that monocyte-mediated gene delivery of IFNα inhibits leukemia in a mouse model. IFN gene therapy counteracts leukemia-induced expansion of immunosuppressive myeloid cells and imposes an immunostimulatory program to the TME, as shown by bulk and single-cell transcriptome analyses. This reprogramming promotes T-cell priming and effector function against multiple surrogate tumor-specific antigens, inhibiting leukemia growth in our experimental model. Durable responses are observed in a fraction of mice and are further increased combining gene therapy with checkpoint blockers. Furthermore, IFN gene therapy strongly enhances anti-tumor activity of adoptively transferred T cells engineered with tumor-specific TCR or CAR, overcoming suppressive signals in the leukemia TME. These findings warrant further investigations on the potential development of our gene therapy strategy towards clinical testing.

An immune suppressive tumor microenvironment (TME) is a limitation for immunotherapy. Here the authors show that, in a B cell acute lymphoblastic leukemia mouse model, gene-based delivery of IFNα  reprograms the leukemia-induced immunosuppressive TME into immunostimulatory and enhances T-cell responses.

• MHC‐I
• gene therapy
• hemophilia
• lentiviral vectors
• stable producer cell line

• fracture repair
• gene therapy
• non-union

• AAV
• CRISPR
• Cas9
• factor IX
• gRNA
• hepatocytes
• liver
• off-target
• truncated gRNA

• Animals
• Base Sequence
• Binding Sites
• CRISPR-Cas Systems
• Computational Biology/methods
• Dependovirus/genetics
• Factor IX/genetics
• Gene Editing
• Gene Targeting
• Genetic Vectors/genetics
• Hemophilia B/diagnosis
• Hemophilia B/genetics
• Hemophilia B/therapy
• Humans
• Liver/metabolism
• Mice
• Organ Specificity
• Phenotype
• Protein Binding
• RNA, Guide, CRISPR-Cas Systems

• Gene therapy
• Immune responses in gene therapy
• Immune-modulation strategies in gene therapy
• Transgene-specific immune tolerance

• CRISPR
• gene editing
• gene therapy
• liver, non-viral vectors
• viral vectors

In lentiviral vector (LV) applications where transient transgene expression is sufficient, integrase-defective lentiviral vectors (IDLVs) are beneficial for reducing the potential for off-target effects associated with insertional mutagenesis. It was previously demonstrated that human RPE65 mRNA expression from an integrating lentiviral vector (ILV) induces endogenous Rpe65 and Cralbp mRNA expression in murine bone marrow–derived cells (BMDCs), initiating programming of the cells to retinal pigment epithelium (RPE)-like cells. These cells regenerate RPE in retinal degeneration models when injected systemically. As transient expression of RPE65 is sufficient to activate endogenous RPE-associated genes for programming BMDCs, use of an ILV is an unnecessary risk. In this study, an IDLV expressing RPE65 (IDLV3-RPE65) was generated. Transduction with IDLV3-RPE65 is less efficient than the integrating vector (ILV3-RPE65). Therefore, IDLV3-RPE65 transduction was enhanced with a combination of preloading 20 × -concentrated viral supernatant on RetroNectin at a multiplicity of infection of 50 and transduction of BMDCs by low-speed centrifugation. RPE65 mRNA levels increased from ∼12-fold to ∼25-fold (p < 0.05) after modification of the IDLV3-RPE65 transduction protocol, achieving expression similar to the ∼27-fold (p < 0.05) increase observed with ILV3-RPE65. Additionally, the study shows that the same preparation of RetroNectin can be used to coat up to three wells with no reduction in transduction. Critically, IDLV3-RPE65 transduction initiates endogenous Rpe65 mRNA expression in murine BMDCs and Cralbp/CRALBP mRNA in both murine and human BMDCs, similar to expression observed in ILV3-RPE65-transduced cells. Systemic administration of ILV3-RPE65 or IDLV3-RPE65 programmed BMDCs in a mouse model of retinal degeneration is sufficient to retain visual function and reduce retinal degeneration compared to mice receiving no treatment or naïve BMDC. It is concluded that IDLV3-RPE65 is appropriate for programming BMDCs to RPE-like cells.

• age-related macular degeneration
• bone marrow–derived cells
• integrase-defective lentivirus
• lentiviral vectors
• retinal pigment epithelium
• transduction efficiency

Hemophilia A (coagulation factor VIII deficiency) is a debilitating genetic disorder that is primarily treated with intravenous replacement therapy. Despite a variety of factor VIII protein formulations available, the risk of developing anti-dug antibodies (“inhibitors”) remains. Overall, 20–30% of patients with severe disease develop inhibitors. Current clinical immune tolerance induction protocols to eliminate inhibitors are not effective in all patients, and there are no prophylactic protocols to prevent the immune response. New experimental therapies, such as gene and cell therapies, show promising results in pre-clinical studies in animal models of hemophilia. Examples include hepatic gene transfer with viral vectors, genetically engineered regulatory T cells (Treg), in vivo Treg induction using immune modulatory drugs, and maternal antigen transfer. Furthermore, an oral tolerance protocol is being developed based on transgenic lettuce plants, which suppressed inhibitor formation in hemophilic mice and dogs. Hopefully, some of these innovative approaches will reduce the risk of and/or more effectively eliminate inhibitor formation in future treatment of hemophilia A.

• AAV vectors
• factor VIII
• gene therapy
• hemophilia A
• immune tolerance
• oral tolerance
• rapamycin
• regulatory T cell

• Tregs
• gene therapy
• hemophilia A
• inhibitor titers reversion
• targeted FVIII expression

The tolerogenic hepatic microenvironment impedes clearance of viral infections but is an advantage in viral vector gene transfer, which often results in immune tolerance induction to transgene products. Although the underlying tolerance mechanism has been extensively studied, our understanding of antigen presentation to transgene product-specific CD4⁺ T cells remains limited. To address this, we administered hepatotropic adeno-associated virus (AAV8) vector expressing cytoplasmic ovalbumin (OVA) into wt mice followed by adoptive transfer of transgenic OVA-specific T cells. We find that that the liver-draining lymph nodes (celiac and portal) are the major sites of MHC II presentation of the virally encoded antigen, as judged by in vivo proliferation of DO11.10 CD4⁺ T cells (requiring professional antigen-presenting cells, e.g., macrophages) and CD4⁺CD25⁺FoxP3⁺ Treg induction. Antigen presentation in the liver itself contributes to activation of CD4⁺ T cells egressing from the liver. Hepatic-induced Treg rapidly disseminate through the systemic circulation. By contrast, a secreted OVA transgene product is presented in multiple organs, and OVA-specific Treg emerge in both the thymus and periphery. In summary, liver draining lymph nodes play an integral role in hepatic antigen presentation and peripheral Treg induction, which results in systemic regulation of the response to viral gene products.

• b cells
• factor IX
• gene therapy
• hemophilia
• lentiviral vector

• Animals
• Antigen-Presenting Cells/cytology
• B-Lymphocytes/cytology
• CD4-Positive T-Lymphocytes/cytology
• Cell Proliferation
• Factor IX/metabolism
• Gene Transfer Techniques
• Genetic Vectors
• HEK293 Cells
• Hemophilia A/blood
• Hemophilia A/therapy
• Humans
• Immunotherapy/methods
• Lentivirus/genetics
• Membrane Glycoproteins/chemistry
• Mice
• Mice, Inbred NOD
• Mice, SCID
• Papio
• Plasmids
• Transduction, Genetic
• Transgenes
• Treatment Outcome
• Viral Envelope Proteins/chemistry

Gene therapy protocols require robust and long-term gene expression. For two decades, retrovirus family vectors have offered several attractive properties as stable gene-delivery vehicles. These vectors represent a technology with widespread use in basic biology and translational studies that require persistent gene expression for treatment of several monogenic diseases. Immunogenicity and insertional mutagenesis represent the main obstacles to a wider clinical use of these vectors. Efficient and safe non-viral vectors are emerging as a promising alternative and facilitate clinical gene therapy studies. Here, we present an updated review for beginners and expert readers on retro and lentiviruses and the latest generation of transposon vectors (sleeping beauty and piggyBac) used in stable gene transfer and gene therapy clinical trials. We discuss the potential advantages and disadvantages of these systems such as cellular responses (immunogenicity or genome modification of the target cell) following exogenous DNA integration. Additionally, we discuss potential implications of these genome modification tools in gene therapy and other basic and applied science contexts.

• Clinical trials
• Gene therapy
• Lentivectors
• Transposons