RESUMO
Genome editing has been a transformative force in the life sciences and human medicine, offering unprecedented opportunities to dissect complex biological processes and treat the underlying causes of many genetic diseases. CRISPR-based technologies, with their remarkable efficiency and easy programmability, stand at the forefront of this revolution. In this Review, we discuss the current state of CRISPR gene editing technologies in both research and therapy, highlighting limitations that constrain them and the technological innovations that have been developed in recent years to address them. Additionally, we examine and summarize the current landscape of gene editing applications in the context of human health and therapeutics. Finally, we outline potential future developments that could shape gene editing technologies and their applications in the coming years.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Disciplinas das Ciências Biológicas , Terapia Genética , TecnologiaRESUMO
Genetic medicines show promise for treating various diseases, yet clinical success has been limited by tolerability, scalability, and immunogenicity issues of current delivery platforms. To overcome these, we developed a proteolipid vehicle (PLV) by combining features from viral and non-viral approaches. PLVs incorporate fusion-associated small transmembrane (FAST) proteins isolated from fusogenic orthoreoviruses into a well-tolerated lipid formulation, using scalable microfluidic mixing. Screening a FAST protein library, we identified a chimeric FAST protein with enhanced membrane fusion activity that improved gene expression from an optimized lipid formulation. Systemically administered FAST-PLVs showed broad biodistribution and effective mRNA and DNA delivery in mouse and non-human primate models. FAST-PLVs show low immunogenicity and maintain activity upon repeat dosing. Systemic administration of follistatin DNA gene therapy with FAST-PLVs raised circulating follistatin levels and significantly increased muscle mass and grip strength. These results demonstrate the promising potential of FAST-PLVs for redosable gene therapies and genetic medicines.
Assuntos
DNA , Proteolipídeos , Animais , Camundongos , DNA/metabolismo , DNA/administração & dosagem , Proteolipídeos/metabolismo , Terapia Genética/métodos , Humanos , Folistatina/metabolismo , Folistatina/genética , Técnicas de Transferência de Genes , RNA/metabolismo , RNA/administração & dosagem , Feminino , Camundongos Endogâmicos C57BLRESUMO
SpCas9 and AsCas12a are widely utilized as genome-editing tools in human cells. However, their relatively large size poses a limitation for delivery by cargo-size-limited adeno-associated virus (AAV) vectors. The type V-F Cas12f from Acidibacillus sulfuroxidans is exceptionally compact (422 amino acids) and has been harnessed as a compact genome-editing tool. Here, we developed an approach, combining deep mutational scanning and structure-informed design, to successfully generate two AsCas12f activity-enhanced (enAsCas12f) variants. Remarkably, the enAsCas12f variants exhibited genome-editing activities in human cells comparable with those of SpCas9 and AsCas12a. The cryoelectron microscopy (cryo-EM) structures revealed that the mutations stabilize the dimer formation and reinforce interactions with nucleic acids to enhance their DNA cleavage activities. Moreover, enAsCas12f packaged with partner genes in an all-in-one AAV vector exhibited efficient knock-in/knock-out activities and transcriptional activation in mice. Taken together, enAsCas12f variants could offer a minimal genome-editing platform for in vivo gene therapy.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Humanos , Camundongos , Microscopia Crioeletrônica , Mutação , Terapia GenéticaRESUMO
Viruses and multinucleated cells rely on fusogens to facilitate the fusion of their membranes. In this issue of Cell, Millay and colleagues demonstrate that replacing viral fusogens with mammalian skeletal muscle fusogens leads to the specific transduction of skeletal muscle and the ability to deliver gene therapy constructs in a therapeutically relevant muscle disease.
Assuntos
Terapia Genética , Músculo Esquelético , Vírus , Animais , Fusão Celular , MamíferosRESUMO
Enhanced by polyamide surfactant Syn3, intravesical administration of rAd-IFNα2b results in transduction of the virus into the bladder epithelium, resulting in the synthesis and expression of local IFNα2b cytokine. Upon secretion, IFNα2b binds to the IFNα receptor on bladder cancer and other cells, resulting in signaling via the JAK-STAT pathway. A plethora of induced IFN-stimulated genes containing IFN-sensitive response elements that contribute to activation of pathways restrict cancer growth.
Assuntos
Janus Quinases , Neoplasias da Bexiga Urinária , Humanos , Fatores de Transcrição STAT , Transdução de Sinais , Adenoviridae , Interferon-alfa , Terapia GenéticaRESUMO
The FDA has recently approved Krystal biotech's beremagene geperpavec (B-VEC, Vyjuvek) to treat the wounds of dystrophic epidermolysis bullosa (DEB) patients. This represents a giant step, not only toward the treatment of this devastating disease, but also for the whole field of non-replicative (nr) recombinant HSV-1 vectors for gene therapy. To view this Bench to Bedside, open or download the PDF.
Assuntos
Epidermólise Bolhosa Distrófica , Terapia Genética , Humanos , Epidermólise Bolhosa Distrófica/genética , Epidermólise Bolhosa Distrófica/terapia , Herpesvirus Humano 1/genéticaRESUMO
CRISPR-Cas9-based base editing allows precise base editing to achieve conversion of adenosine to guanine or cytosine to thymidine. In this issue of Cell, McAuley et al. use adenine base editing to correct a single base-pair mutation causing human CD3δ deficiency, demonstrating superior efficiency of genetic correction with reduced undesired genetic alterations compared with standard CRISPR-Cas9 editing.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Doenças do Sistema Imunitário , Humanos , Adenina , Sistemas CRISPR-Cas/genética , Terapia Genética , Mutação , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/terapiaRESUMO
Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans.
Assuntos
Terapia Genética , Doença de Parkinson , Animais , Humanos , Camundongos , Corpo Estriado/metabolismo , Levodopa/uso terapêutico , Levodopa/genética , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/terapia , Primatas , Receptores de Dopamina D1/metabolismo , Modelos Animais de DoençasRESUMO
In vivo gene editing therapies offer the potential to treat the root causes of many genetic diseases. Realizing the promise of therapeutic in vivo gene editing requires the ability to safely and efficiently deliver gene editing agents to relevant organs and tissues in vivo. Here, we review current delivery technologies that have been used to enable therapeutic in vivo gene editing, including viral vectors, lipid nanoparticles, and virus-like particles. Since no single delivery modality is likely to be appropriate for every possible application, we compare the benefits and drawbacks of each method and highlight opportunities for future improvements.
Assuntos
Edição de Genes , Nanopartículas , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Terapia Genética/métodos , Vetores Genéticos , LipossomosRESUMO
Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) is proving successful to treat several genetic diseases. HSPCs are mobilized, harvested, genetically corrected ex vivo, and infused, after the administration of toxic myeloablative conditioning to deplete the bone marrow (BM) for the modified cells. We show that mobilizers create an opportunity for seamless engraftment of exogenous cells, which effectively outcompete those mobilized, to repopulate the depleted BM. The competitive advantage results from the rescue during ex vivo culture of a detrimental impact of mobilization on HSPCs and can be further enhanced by the transient overexpression of engraftment effectors exploiting optimized mRNA-based delivery. We show the therapeutic efficacy in a mouse model of hyper IgM syndrome and further developed it in human hematochimeric mice, showing its applicability and versatility when coupled with gene transfer and editing strategies. Overall, our findings provide a potentially valuable strategy paving the way to broader and safer use of HSPC-GT.
Assuntos
Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Animais , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas , Humanos , CamundongosRESUMO
Through studies in mice and in humans, Stuart Orkin showed that GATA-1 is a master transcriptional regulator of hematopoiesis. He has highlighted the role of BCL11A in the fetal-adult hemoglobin switch. The Gairdner Foundation Award recognizes Orkin's contribution to the development of gene therapy of sickle cell disease.
Assuntos
Anemia Falciforme , Distinções e Prêmios , Terapia Genética , Anemia Falciforme/genética , Anemia Falciforme/terapia , Animais , Modelos Animais de Doenças , Hemoglobina Fetal/genética , Hematopoese/genética , Humanos , Camundongos , Proteínas Repressoras/genéticaRESUMO
Adoptive immunotherapy, or the infusion of lymphocytes, is a promising approach for the treatment of cancer and certain chronic viral infections. The application of the principles of synthetic biology to enhance T cell function has resulted in substantial increases in clinical efficacy. The primary challenge to the field is to identify tumor-specific targets to avoid off-tumor, on-target toxicity. Given recent advances in efficacy in numerous pilot trials, the next steps in clinical development will require multicenter trials to establish adoptive immunotherapy as a mainstream technology.
Assuntos
Imunoterapia Adotiva , Neoplasias/imunologia , Neoplasias/terapia , Viroses/imunologia , Viroses/terapia , Transferência Adotiva , Animais , Antígenos/genética , Antígenos/imunologia , Biomarcadores , Terapia Baseada em Transplante de Células e Tecidos , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Neoplasias/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transdução Genética , Viroses/genéticaRESUMO
The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-based biotechnologies has revolutionized the life sciences and introduced new therapeutic modalities with the potential to treat a wide range of diseases. Here, we describe CRISPR-based strategies to improve human health, with an emphasis on the delivery of CRISPR therapeutics directly into the human body using adeno-associated virus (AAV) vectors. We also discuss challenges facing broad deployment of CRISPR-based therapeutics and highlight areas where continued discovery and technological development can further advance these revolutionary new treatments.
Assuntos
Sistemas CRISPR-Cas , Dependovirus/genética , Edição de Genes/métodos , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Animais , HumanosRESUMO
Mitochondrial diseases are clinically heterogeneous disorders caused by a wide spectrum of mutations in genes encoded by either the nuclear or the mitochondrial genome. Treatments for mitochondrial diseases are currently focused on symptomatic management rather than improving the biochemical defect caused by a particular mutation. This review focuses on the latest advances in the development of treatments for mitochondrial disease, both small molecules and gene therapies, as well as methods to prevent transmission of mitochondrial disease through the germline.
Assuntos
Mitocôndrias/genética , Doenças Mitocondriais/terapia , Animais , DNA Mitocondrial/genética , Terapia Genética , Genoma Mitocondrial/genética , Humanos , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Mutação , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Tazemetostat is the first epigenetic therapy to gain FDA approval in a solid tumor. This lysine methyltransferase inhibitor targets EZH2, the enzymatic subunit of the PRC2 transcriptional silencing complex. Tumors with mutations in subunits of the SWI/SNF chromatin remodeling complex, inclusive of most epithelioid sarcomas, are sensitive to EZH2 inhibition.
Assuntos
Benzamidas/uso terapêutico , Epigênese Genética/genética , Piridonas/uso terapêutico , Sarcoma/tratamento farmacológico , Compostos de Bifenilo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Inibidores Enzimáticos/farmacologia , Epigenômica , Terapia Genética/métodos , Humanos , Morfolinas , Proteínas Nucleares/metabolismo , Sarcoma/genética , Fatores de Transcrição/metabolismoRESUMO
Many targeted base transversions, insertions, and deletions remain challenging due to the lack of precise and efficient genome editing technologies. Recently, Anzalone et al. reported a versatile approach to achieve all types of genome edits, shedding new light on correcting most genetic variants associated with diseases.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Animais , Terapia Genética/métodos , HumanosRESUMO
The power of CRISPR is undeniable, but where is the field heading? Cell's April Pawluk caught up with Jia Chen, Weizhi Ji, and Prashant Mali to discuss the successes and challenges we can expect in the coming years. Annotated excerpts from this conversation are presented below, and the full conversation is available with the article online.
Assuntos
Edição de Genes/métodos , Edição de Genes/tendências , Terapia Genética/métodos , Terapia Genética/tendências , Genoma Humano , Animais , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , HumanosRESUMO
Biallelic mutations in the RPE65 gene are associated with inherited retinal degenerations/dystrophies (IRD) and disrupt the visual cycle, leading to loss of vision. A new adenoviral vector-based gene therapy surgically delivered to retinal cells provides normal human RPE65 protein that can restore the visual cycle and some vision. To view this Bench to Bedside, open or download the PDF.
Assuntos
Degeneração Retiniana/terapia , Adenoviridae/genética , Terapia Genética , Vetores Genéticos/economia , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Humanos , Amaurose Congênita de Leber/epidemiologia , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/terapia , Degeneração Retiniana/epidemiologia , Degeneração Retiniana/genética , cis-trans-Isomerases/genética , cis-trans-Isomerases/metabolismoRESUMO
The CRISPR-Cas9 RNA-guided DNA endonuclease has contributed to an explosion of advances in the life sciences that have grown from the ability to edit genomes within living cells. In this Review, we summarize CRISPR-based technologies that enable mammalian genome editing and their various applications. We describe recent developments that extend the generality, DNA specificity, product selectivity, and fundamental capabilities of natural CRISPR systems, and we highlight some of the remarkable advancements in basic research, biotechnology, and therapeutics science that these developments have facilitated.
Assuntos
Edição de Genes/métodos , Animais , Sistemas CRISPR-Cas , Células Cultivadas , Doença/genética , Modelos Animais de Doenças , Epigenômica/métodos , Terapia Genética , HumanosRESUMO
A phase 1/2 clinical trial of AAV-mediated gene therapy in patients with hemophilia B using an enhanced specific activity factor IX (FIX) transgene reports sustained levels of FIX levels, leading to the near elimination of bleeding for more than a year and without serious adverse side effects. These results are the best outcome to date for hemophilia gene therapy.