ABSTRACT
ABSTRACT: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo. Here, we describe the incorporation of T-cell activation and costimulatory signals onto the surface of VivoVec particles (VVPs) in the form of a multidomain fusion protein and show enhanced in vivo transduction and improved CAR T-cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into nonhuman primates resulted in the robust generation of anti-CD20 CAR T cells and the complete depletion of B cells for >10 weeks. These data validate the VivoVec platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.
Subject(s)
Genetic Vectors , Immunotherapy, Adoptive , Lentivirus , Receptors, Chimeric Antigen , T-Lymphocytes , Animals , Lentivirus/genetics , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Humans , Immunotherapy, Adoptive/methods , Ligands , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Transduction, Genetic , Antigens, CD20/immunology , Antigens, CD20/genetics , Lymphocyte ActivationABSTRACT
Engineered T cells represent an emerging therapeutic modality. However, complex engineering strategies can present a challenge for enriching and expanding therapeutic cells at clinical scale. In addition, lack of in vivo cytokine support can lead to poor engraftment of transferred T cells, including regulatory T cells (Treg). Here, we establish a cell-intrinsic selection system that leverages the dependency of primary T cells on IL-2 signaling. FRB-IL2RB and FKBP-IL2RG fusion proteins were identified permitting selective expansion of primary CD4+ T cells in rapamycin supplemented medium. This chemically inducible signaling complex (CISC) was subsequently incorporated into HDR donor templates designed to drive expression of the Treg master regulator FOXP3. Following editing of CD4+ T cells, CISC+ engineered Treg (CISC EngTreg) were selectively expanded using rapamycin and maintained Treg activity. Following transfer into immunodeficient mice treated with rapamycin, CISC EngTreg exhibited sustained engraftment in the absence of IL-2. Furthermore, in vivo CISC engagement increased the therapeutic activity of CISC EngTreg. Finally, an editing strategy targeting the TRAC locus permitted generation and selective enrichment of CISC+ functional CD19-CAR-T cells. Together, CISC provides a robust platform to achieve both in vitro enrichment and in vivo engraftment and activation, features likely beneficial across multiple gene-edited T cell applications.
Subject(s)
CD4-Positive T-Lymphocytes , Interleukin-2 , Mice , Animals , CD4-Positive T-Lymphocytes/metabolism , Interleukin-2/genetics , Interleukin-2/pharmacology , Interleukin-2/metabolism , T-Lymphocytes, Regulatory/metabolism , Sirolimus/pharmacology , Receptors, Interleukin-2/metabolismABSTRACT
BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation. METHODS: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice. RESULTS: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy. CONCLUSIONS: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.
Subject(s)
Receptors, Chimeric Antigen , T-Lymphocytes , Humans , Animals , Dogs , Mice , Receptors, Chimeric Antigen/genetics , Receptors, Antigen, T-Cell , Leukocytes, Mononuclear , Tissue Distribution , Cell Engineering/methodsABSTRACT
Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a "GG" dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases.
Subject(s)
CRISPR-Associated Protein 9/genetics , CRISPR-Cas Systems/genetics , Gene Editing/methods , Staphylococcus/enzymology , Animals , CRISPR-Associated Protein 9/isolation & purification , Cell Line, Tumor , Dependovirus , Disease Models, Animal , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , HEK293 Cells , Humans , Macaca fascicularis , Male , Mice , Parvovirinae/genetics , Protein Engineering , Ribonucleases , Staphylococcus/genetics , Substrate Specificity , Usher Syndromes/genetics , Usher Syndromes/therapy , RNA, Guide, CRISPR-Cas SystemsABSTRACT
Thymic regulatory T cells (tTregs) are potent inhibitors of autoreactive immune responses, and loss of tTreg function results in fatal autoimmune disease. Defects in tTreg number or function are also implicated in multiple autoimmune diseases, leading to growing interest in use of Treg as cell therapies to establish immune tolerance. Because tTregs are present at low numbers in circulating blood and may be challenging to purify and expand and also inherently defective in some subjects, we designed an alternative strategy to create autologous Treg-like cells from bulk CD4+ T cells. We used homology-directed repair (HDR)-based gene editing to enforce expression of FOXP3, the master transcription factor for tTreg Targeted insertion of a robust enhancer/promoter proximal to the first coding exon bypassed epigenetic silencing, permitting stable and robust expression of endogenous FOXP3. HDR-edited T cells, edTregs, manifested a transcriptional program leading to sustained expression of canonical markers and suppressive activity of tTreg Both human and murine edTregs mediated immunosuppression in vivo in models of inflammatory disease. Further, this engineering strategy permitted generation of antigen-specific edTreg with robust in vitro and in vivo functional activity. Last, edTreg could be enriched and expanded at scale using clinically relevant methods. Together, these findings suggest that edTreg production may permit broad future clinical application.
Subject(s)
Forkhead Transcription Factors , Gene Editing , Animals , Forkhead Transcription Factors/genetics , Humans , Immune Tolerance , Mice , Phenotype , T-Lymphocytes, RegulatoryABSTRACT
Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34+CD90+CD45RA-, allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.
Subject(s)
CRISPR-Cas Systems/genetics , Fetal Hemoglobin/metabolism , Hematopoietic Stem Cells/cytology , Animals , Antigens, CD34/metabolism , Fetal Hemoglobin/genetics , Gene Editing , Genotype , Hematopoietic Stem Cell Transplantation , Humans , Macaca mulatta , Primates , Thy-1 Antigens/metabolismABSTRACT
Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34+ mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro, rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12-14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ-/- Kit(W41/W41) (NBSGW) mice, a â¼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSCs. Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation.
ABSTRACT
Elements within the γ-hemoglobin promoters (HBG1 and HBG2) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the HBG1 promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous HBG promoters to de-repress fetal hemoglobin. Transfection of human CD34+ cells with TALEN mRNA resulted in indel generation in HBG1 (43%) and HBG2 (74%) including the 13-bp HPFH deletion (â¼6%). Erythroid differentiation of edited cells revealed a 4.6-fold increase in γ-hemoglobin expression as detected by HPLC. Assessment of TALEN-edited CD34+ cells in vivo in a humanized mouse model demonstrated sustained presence of indels in hematopoietic cells up to 24 weeks. Indel rates remained unchanged following secondary transplantation consistent with editing of long-term repopulating stem cells (LT-HSCs). Human γ-hemoglobin expressing F cells were detected by flow cytometry approximately 50% more frequently in edited animals compared to mock. Together, these findings demonstrate that TALEN-mediated indel generation in the γ-hemoglobin promoter leads to high levels of fetal hemoglobin expression in vitro and in vivo, suggesting that this approach can provide therapeutic benefit in patients with SCD or ß-thalassemia.
ABSTRACT
Hematopoietic stem-cell gene therapy is a promising treatment of X-linked severe combined immunodeficiency disease (SCID-X1), but currently, it requires recipient conditioning, extensive cell manipulation, and sophisticated facilities. With these limitations in mind, we explored a simpler therapeutic approach to SCID-X1 treatment by direct IV administration of foamy virus (FV) vectors in the canine model. FV vectors were used because they have a favorable integration site profile and are resistant to serum inactivation. Here, we show improved efficacy of our in vivo gene therapy platform by mobilization with granulocyte colony-stimulating factor (G-CSF) and AMD3100 before injection of an optimized FV vector incorporating the human phosphoglycerate kinase enhancerless promoter. G-CSF/AMD3100 mobilization before FV vector delivery accelerated kinetics of CD3+ lymphocyte recovery, promoted thymopoiesis, and increased immune clonal diversity. Gene-corrected T lymphocytes exhibited a normal CD4:CD8 ratio and a broad T-cell receptor repertoire and showed restored γC-dependent signaling function. Treated animals showed normal primary and secondary antibody responses to bacteriophage immunization and evidence for immunoglobulin class switching. These results demonstrate safety and efficacy of an accessible, portable, and translatable platform with no conditioning regimen for the treatment of SCID-X1 and other genetic diseases.
Subject(s)
Dog Diseases , Genetic Therapy , Genetic Vectors/pharmacology , Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cell Mobilization , Heterocyclic Compounds/pharmacology , Spumavirus , X-Linked Combined Immunodeficiency Diseases , Animals , Benzylamines , CD4-CD8 Ratio , Cyclams , Disease Models, Animal , Dog Diseases/blood , Dog Diseases/genetics , Dog Diseases/therapy , Dogs , Humans , Phosphoglycerate Kinase/genetics , X-Linked Combined Immunodeficiency Diseases/blood , X-Linked Combined Immunodeficiency Diseases/genetics , X-Linked Combined Immunodeficiency Diseases/therapy , X-Linked Combined Immunodeficiency Diseases/veterinaryABSTRACT
[This corrects the article DOI: 10.1371/journal.pbio.1000428.].
ABSTRACT
Therapeutic gene editing is significant for medical advancement. Safety is intricately linked to the specificity of the editing tools used to cut at precise genomic targets. Improvements can be achieved by thoughtful design of nucleases and repair templates, analysis of off-target editing, and careful utilization of viral vectors. Advancements in DNA repair mechanisms and development of new generations of tools improve targeting of specific sequences while minimizing risks. It is important to plot a safe course for future clinical trials. This article reviews safety and specificity for therapeutic gene editing to spur dialogue and advancement.
Subject(s)
Gene Editing , Genetic Therapy , Genetic Vectors/genetics , Animals , DNA Repair , Gene Editing/methods , Gene Targeting , Gene Transfer Techniques , Genetic Therapy/adverse effects , Genetic Therapy/methods , Homologous Recombination , Humans , Transduction, GeneticABSTRACT
Gene editing by homology-directed recombination (HDR) can be used to couple delivery of a therapeutic gene cassette with targeted genomic modifications to generate engineered human T cells with clinically useful profiles. Here, we explore the functionality of therapeutic cassettes delivered by these means and test the flexibility of this approach to clinically relevant alleles. Because CCR5-negative T cells are resistant to HIV-1 infection, CCR5-negative anti-CD19 chimeric antigen receptor (CAR) T cells could be used to treat patients with HIV-associated B cell malignancies. We show that targeted delivery of an anti-CD19 CAR cassette to the CCR5 locus using a recombinant AAV homology template and an engineered megaTAL nuclease results in T cells that are functionally equivalent, in both in vitro and in vivo tumor models, to CAR T cells generated by random integration using lentiviral delivery. With the goal of developing off-the-shelf CAR T cell therapies, we next targeted CARs to the T cell receptor alpha constant (TRAC) locus by HDR, producing TCR-negative anti-CD19 CAR and anti-B cell maturation antigen (BCMA) CAR T cells. These novel cell products exhibited in vitro cytolytic activity against both tumor cell lines and primary cell targets. Our combined results indicate that high-efficiency HDR delivery of therapeutic genes may provide a flexible and robust method that can extend the clinical utility of cell therapeutics.
ABSTRACT
BACKGROUND: Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase-deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. METHODS: Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) γ-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. RESULTS: With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1-2.6) and granulocytes (VCN = 0.01-0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. CONCLUSION: These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. TRIAL REGISTRATION: ClinicalTrials.gov NCT00794508. FUNDING: Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA Clinical and Translational Science Institute awards, UL1RR033176 and UL1TR000124.
Subject(s)
Adenosine Deaminase/deficiency , Agammaglobulinemia , Gene Expression Regulation, Enzymologic , Genetic Therapy , Hematopoietic Stem Cell Transplantation , Severe Combined Immunodeficiency , Transduction, Genetic , Adenosine Deaminase/biosynthesis , Adenosine Deaminase/genetics , Adolescent , Agammaglobulinemia/enzymology , Agammaglobulinemia/genetics , Agammaglobulinemia/therapy , Autografts , Child , Child, Preschool , Female , Genetic Vectors , Humans , Infant , Male , Retroviridae , Severe Combined Immunodeficiency/enzymology , Severe Combined Immunodeficiency/genetics , Severe Combined Immunodeficiency/therapyABSTRACT
LAGLIDADG homing endonucleases (LHEs) are a class of rare-cleaving nucleases that possess several unique attributes for genome engineering applications. An important approach for advancing LHE technology is the generation of a library of design 'starting points' through the discovery and characterization of natural LHEs with diverse specificities. However, while identification of natural LHE proteins by sequence homology from genomic and metagenomic sequence databases is straightforward, prediction of corresponding target sequences from genomic data remains challenging. Here, we describe a general approach that we developed to circumvent this issue that combines two technologies: yeast surface display (YSD) of LHEs and systematic evolution of ligands via exponential enrichment (SELEX). Using LHEs expressed on the surface of yeast, we show that SELEX can yield binding specificity motifs and identify cleavable LHE targets using a combination of bioinformatics and biochemical cleavage assays. This approach, which we term YSD-SELEX, represents a simple and rapid first principles approach to determining the binding and cleavage specificity of novel LHEs that should also be generally applicable to any type of yeast surface expressible DNA-binding protein. In this marriage, SELEX adds DNA specificity determination to the YSD platform, and YSD brings diagnostics and inexpensive, facile protein-matrix generation to SELEX.
Subject(s)
Endonucleases/metabolism , SELEX Aptamer Technique/methods , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Motifs , Amino Acid Sequence , Catalytic Domain/genetics , DNA, Fungal/genetics , DNA, Fungal/metabolism , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/metabolism , Endonucleases/chemistry , Endonucleases/genetics , Phylogeny , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
The treatment or cure of HIV infection by cell and gene therapy has been a goal for decades. Recent advances in both gene editing and chimeric antigen receptor (CAR) technology have created new therapeutic possibilities for a variety of diseases. Broadly neutralizing monoclonal antibodies (bNAbs) with specificity for the HIV envelope glycoprotein provide a promising means of targeting HIV-infected cells. Here we show that primary human T cells engineered to express anti-HIV CARs based on bNAbs (HIVCAR) show specific activation and killing of HIV-infected versus uninfected cells in the absence of HIV replication. We also show that homology-directed recombination of the HIVCAR gene expression cassette into the CCR5 locus enhances suppression of replicating virus compared with HIVCAR expression alone. This work demonstrates that HIV immunotherapy utilizing potent bNAb-based single-chain variable fragments fused to second-generation CAR signaling domains, delivered directly into the CCR5 locus of T cells by homology-directed gene editing, is feasible and effective. This strategy has the potential to target HIV-infected cells in HIV-infected individuals, which might help in the effort to cure HIV.
Subject(s)
HIV Antibodies/immunology , HIV Infections/immunology , HIV Infections/therapy , HIV-1/immunology , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Receptors, Antigen, T-Cell/metabolism , Recombinant Fusion Proteins , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Cytotoxicity, Immunologic , Epitopes/immunology , Gene Order , Genetic Engineering , Genetic Vectors/genetics , HIV Antibodies/genetics , HIV Antibodies/metabolism , HIV Envelope Protein gp120/immunology , HIV Infections/genetics , Humans , Immunotherapy , Lymphocyte Activation/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, CCR5/genetics , Receptors, CCR5/metabolism , Single-Chain Antibodies , Virus ReplicationABSTRACT
Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components-Cas9, guide RNAs and recombination templates-to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Mutant Proteins , T-Lymphocytes/metabolism , Viral Proteins/metabolism , Dependovirus/genetics , Gene Expression , Gene Knock-In Techniques , Gene Knockout Techniques , Gene Order , Gene Targeting , Gene Transfer Techniques , Genetic Vectors/genetics , Homologous Recombination , Humans , RNA, Guide, Kinetoplastida/genetics , Transduction, Genetic , Viral Proteins/geneticsABSTRACT
LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity.
Subject(s)
DNA/chemistry , DNA/metabolism , Deoxyribonucleases/chemistry , Deoxyribonucleases/metabolism , Base Sequence , Binding Sites , DNA Cleavage , Models, Molecular , Nucleic Acid Conformation , Protein Conformation , Substrate SpecificityABSTRACT
Gene editing is a rapidly developing area of biotechnology in which the nucleotide sequence of the genome of living cells is precisely changed. The use of genome-editing technologies to modify various types of blood cells, including hematopoietic stem cells, has emerged as an important field of therapeutic development for hematopoietic disease. Although these technologies offer the potential for generation of transformative therapies for patients suffering from myriad disorders of hematopoiesis, their application for therapeutic modification of primary human cells is still in its infancy. Consequently, development of ethical and regulatory frameworks that ensure their safe and effective use is an increasingly important consideration. Here, we review a number of issues that have the potential to impact the clinical implementation of genome-editing technologies, and suggest paths forward for resolving them such that new therapies can be safely and rapidly translated to the clinic.
Subject(s)
Bioethical Issues , Gene Editing , Animals , Gene Editing/ethics , Gene Editing/legislation & jurisprudence , Gene Editing/methods , Humans , Targeted Gene Repair/ethics , Targeted Gene Repair/legislation & jurisprudence , Targeted Gene Repair/methodsABSTRACT
Increasing demand for large-scale synthesis of in vitro transcribed (IVT) mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3' polyadenosine (poly(A)) tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A) tail lengths to ~120 base pairs (bp). Here, we have developed a novel method for generation of extended poly(A) tracts using a previously described linear plasmid system, pJazz. We find that linear plasmids can successfully propagate poly(A) tracts up to ~500 bp in length for IVT mRNA production. We then modified pJazz by removing extraneous restriction sites, adding a T7 promoter sequence upstream from an extended multiple cloning site, and adding a unique type-IIS restriction site downstream from the encoded poly(A) tract to facilitate generation of IVT mRNA with precisely defined encoded poly(A) tracts and 3' termini. The resulting plasmid, designated pEVL, can be used to generate IVT mRNA with consistent defined lengths and terminal residue(s).