RESUMO
Entry of enveloped viruses into cells is mediated by viral fusogenic proteins that drive membrane rearrangements needed for fusion between viral and target membranes. Skeletal muscle development also requires membrane fusion events between progenitor cells to form multinucleated myofibers. Myomaker and Myomerger are muscle-specific cell fusogens but do not structurally or functionally resemble classical viral fusogens. We asked whether the muscle fusogens could functionally substitute for viral fusogens, despite their structural distinctiveness, and fuse viruses to cells. We report that engineering of Myomaker and Myomerger on the membrane of enveloped viruses leads to specific transduction of skeletal muscle. We also demonstrate that locally and systemically injected virions pseudotyped with the muscle fusogens can deliver µDystrophin to skeletal muscle of a mouse model of Duchenne muscular dystrophy and alleviate pathology. Through harnessing the intrinsic properties of myogenic membranes, we establish a platform for delivery of therapeutic material to skeletal muscle.
Assuntos
Bioengenharia , Lentivirus , Proteínas de Membrana , Músculo Esquelético , Distrofia Muscular de Duchenne , Animais , Camundongos , Fusão Celular , Fusão de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/virologia , Bioengenharia/métodos , Distrofia Muscular de Duchenne/terapia , Modelos Animais de Doenças , Tropismo Viral , Lentivirus/genéticaRESUMO
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
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
Replacing or editing disease-causing mutations holds great promise for treating many human diseases. Yet, delivering therapeutic genetic modifiers to specific cells in vivo has been challenging, particularly in large, anatomically distributed tissues such as skeletal muscle. Here, we establish an in vivo strategy to evolve and stringently select capsid variants of adeno-associated viruses (AAVs) that enable potent delivery to desired tissues. Using this method, we identify a class of RGD motif-containing capsids that transduces muscle with superior efficiency and selectivity after intravenous injection in mice and non-human primates. We demonstrate substantially enhanced potency and therapeutic efficacy of these engineered vectors compared to naturally occurring AAV capsids in two mouse models of genetic muscle disease. The top capsid variants from our selection approach show conserved potency for delivery across a variety of inbred mouse strains, and in cynomolgus macaques and human primary myotubes, with transduction dependent on target cell expressed integrin heterodimers.
Assuntos
Capsídeo/metabolismo , Dependovirus/metabolismo , Evolução Molecular Direcionada , Técnicas de Transferência de Genes , Músculo Esquelético/metabolismo , Sequência de Aminoácidos , Animais , Capsídeo/química , Células Cultivadas , Modelos Animais de Doenças , Células HEK293 , Humanos , Integrinas/metabolismo , Macaca fascicularis , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/terapia , Multimerização Proteica , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/uso terapêutico , RNA Guia de Cinetoplastídeos/metabolismo , Recombinação Genética/genética , Especificidade da Espécie , TransgenesRESUMO
Epitranscriptomic regulation controls information flow through the central dogma and provides unique opportunities for manipulating cells at the RNA level. However, both fundamental studies and potential translational applications are impeded by a lack of methods to target specific RNAs with effector proteins. Here, we present CRISPR-Cas-inspired RNA targeting system (CIRTS), a protein engineering strategy for constructing programmable RNA control elements. We show that CIRTS is a simple and generalizable approach to deliver a range of effector proteins, including nucleases, degradation machinery, translational activators, and base editors to target transcripts. We further demonstrate that CIRTS is not only smaller than naturally occurring CRISPR-Cas programmable RNA binding systems but can also be built entirely from human protein parts. CIRTS provides a platform to probe fundamental RNA regulatory processes, and the human-derived nature of CIRTS provides a potential strategy to avoid immune issues when applied to epitranscriptome-modulating therapies.
Assuntos
Edição de Genes/métodos , Engenharia de Proteínas/métodos , RNA Guia de Cinetoplastídeos/metabolismo , RNA/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Sistemas CRISPR-Cas/genética , Escherichia coli/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Biossíntese de Proteínas , Proteólise , RNA Interferente Pequeno , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , TransfecçãoRESUMO
The cochlear implant (CI) is considered the most successful neuroprosthesis as it enables speech comprehension in the majority of the million otherwise deaf patients. In hearing by electrical stimulation of the auditory nerve, the broad spread of current from each electrode acts as a bottleneck that limits the transfer of sound frequency information. Hence, there remains a major unmet medical need for improving the quality of hearing with CIs. Recently, optogenetic stimulation of the cochlea has been suggested as an alternative approach for hearing restoration. Cochlear optogenetics promises to transfer more sound frequency information, hence improving hearing, as light can conveniently be confined in space to activate the auditory nerve within smaller tonotopic ranges. In this review, we discuss the latest experimental and technological developments of optogenetic hearing restoration and outline remaining challenges en route to clinical translation.
Assuntos
Implantes Cocleares , Optogenética , Optogenética/métodos , Humanos , Animais , Perda Auditiva/fisiopatologia , Audição/fisiologia , Cóclea/fisiologiaRESUMO
Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Terapia Genética/métodos , Oligonucleotídeos Antissenso/farmacologia , Animais , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Repetições de Microssatélites , Splicing de RNA , Expansão das Repetições de TrinucleotídeosRESUMO
Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Edição de Genes/métodos , Inativação Gênica , Marcação de Genes/métodos , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Repressoras/metabolismo , Domínio Catalítico , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , DNA Metiltransferase 3A , Células-Tronco Embrionárias/metabolismo , Engenharia Genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Proteínas Repressoras/genética , Linfócitos T/metabolismoRESUMO
Salivary glands produce and secrete saliva, which is essential for maintaining oral health and overall health. Understanding both the unique structure and physiological function of salivary glands, as well as how they are affected by disease and injury, will direct the development of therapy to repair and regenerate them. Significant recent advances, particularly in the OMICS field, increase our understanding of how salivary glands develop at the cellular, molecular, and genetic levels: the signaling pathways involved, the dynamics of progenitor cell lineages in development, homeostasis, and regeneration, and the role of the extracellular matrix microenvironment. These provide a template for cell and gene therapies as well as bioengineering approaches to repair or regenerate salivary function.
Assuntos
Regeneração , Glândulas Salivares , Linhagem da Célula , Humanos , Saúde Bucal , Regeneração/fisiologia , Glândulas Salivares/fisiologia , Transdução de SinaisRESUMO
Multiple sclerosis (MS) is a demyelinating, autoimmune disease of the central nervous system. While work has focused on myelin and axon loss in MS, less is known about mechanisms underlying synaptic changes. Using postmortem human MS tissue, a preclinical nonhuman primate model of MS, and two rodent models of demyelinating disease, we investigated synapse changes in the visual system. Similar to other neurodegenerative diseases, microglial synaptic engulfment and profound synapse loss were observed. In mice, synapse loss occurred independently of local demyelination and neuronal degeneration but coincided with gliosis and increased complement component C3, but not C1q, at synapses. Viral overexpression of the complement inhibitor Crry at C3-bound synapses decreased microglial engulfment of synapses and protected visual function. These results indicate that microglia eliminate synapses through the alternative complement cascade in demyelinating disease and identify a strategy to prevent synapse loss that may be broadly applicable to other neurodegenerative diseases. VIDEO ABSTRACT.
Assuntos
Complemento C3/imunologia , Encefalomielite Autoimune Experimental/patologia , Microglia/patologia , Esclerose Múltipla/patologia , Sinapses/patologia , Tálamo/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Callithrix , Linhagem Celular Tumoral , Complemento C3/antagonistas & inibidores , Modelos Animais de Doenças , Feminino , Gliose/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptores de Complemento 3b/metabolismoRESUMO
Long-term delivery of anti-HIV monoclonal antibodies (mAbs) using adeno-associated virus (AAV) vectors holds promise for the prevention and treatment of HIV infection. We describe a therapy trial in which four rhesus monkeys were infected with SHIV-AD8 for 86 weeks before receiving the AAV-encoded mAbs 3BNC117, 10-1074, and 10E8. Although anti-drug antibody (ADA) responses restricted mAb delivery, one monkey successfully maintained 50-150 µg/mL of 3BNC117 and 10-1074 for over 2 years. Delivery of these two mAbs to this monkey resulted in an abrupt decline in plasma viremia, which remained undetectable for 38 successive measurements over 3 years. We generated two more examples of virologic suppression using AAV delivery of a cocktail of four mAbs in a 12-monkey study. Our results provide proof of concept for AAV-delivered mAbs to produce a "functional cure." However, they also serve as a warning that ADAs may be a problem for practical application of this approach in humans.
Assuntos
Anticorpos Monoclonais/imunologia , Dependovirus/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Animais , Anticorpos Monoclonais Humanizados , Anticorpos Neutralizantes/imunologia , Anticorpos Amplamente Neutralizantes , Linhagem Celular , Células HEK293 , Anticorpos Anti-HIV/imunologia , Humanos , Macaca mulatta , Viremia/imunologiaRESUMO
FOXP3 gene is a key transcription factor driving immune tolerance and its deficiency causes immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome (IPEX), a prototypic primary immune regulatory disorder (PIRD) with defective regulatory T (Treg) cells. Although life-threatening, the increased awareness and early diagnosis have contributed to improved control of the disease. IPEX currently comprises a broad spectrum of clinical autoimmune manifestations from severe early onset organ involvement to moderate, recurrent manifestations. This review focuses on the mechanistic advancements that, since the IPEX discovery in early 2000, have informed the role of the human FOXP3+ Treg cells in controlling peripheral tolerance and shaping the overall immune landscape of IPEX patients and carrier mothers, contributing to defining new treatments.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X , Doenças do Sistema Imunitário , Enteropatias , Poliendocrinopatias Autoimunes , Humanos , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Linfócitos T Reguladores , Enteropatias/genética , Síndrome , Fatores de Transcrição Forkhead/genética , Mutação , Poliendocrinopatias Autoimunes/genética , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/terapiaRESUMO
Inborn errors of immunity (IEI) present a unique paradigm in the realm of gene therapy, emphasizing the need for precision in therapeutic design. As gene therapy transitions from broad-spectrum gene addition to careful modification of specific genes, the enduring safety and effectiveness of these therapies in clinical settings have become crucial. This review discusses the significance of IEIs as foundational models for pioneering and refining precision medicine. We explore the capabilities of gene addition and gene correction platforms in modifying the DNA sequence of primary cells tailored for IEIs. The review uses four specific IEIs to highlight key issues in gene therapy strategies: X-linked agammaglobulinemia (XLA), X-linked chronic granulomatous disease (X-CGD), X-linked hyper IgM syndrome (XHIGM), and immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX). We detail the regulatory intricacies and therapeutic innovations for each disorder, incorporating insights from relevant clinical trials. For most IEIs, regulated expression is a vital aspect of the underlying biology, and we discuss the importance of endogenous regulation in developing gene therapy strategies.
Assuntos
Agamaglobulinemia , Doenças Genéticas Ligadas ao Cromossomo X , Enteropatias , Humanos , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Enteropatias/genética , Enteropatias/terapia , Agamaglobulinemia/genética , Agamaglobulinemia/terapia , Terapia GenéticaRESUMO
Severe combined immune deficiency due to adenosine deaminase deficiency (ADA SCID) is an inborn error of immunity with pan-lymphopenia, due to accumulated cytotoxic adenine metabolites. ADA SCID has been treated using gene therapy with a normal human ADA gene added to autologous hematopoietic stem cells (HSC) for over 30 years. Iterative improvements in vector design, HSC processing methods, and clinical HSC transplant procedures have led nearly all ADA SCID gene therapy patients to achieve consistently beneficial immune restoration with stable engraftment of ADA gene-corrected HSC over the duration of observation (as long as 20 years). One gene therapy for ADA SCID is approved by the European Medicines Agency (EMA) in the European Union (EU) and another is being advanced to licensure in the U.S. and U.K. Despite the clear-cut benefits and safety of this curative gene and cell therapy, it remains challenging to achieve sustained availability and access, especially for rare disorders like ADA SCID.
Assuntos
Agamaglobulinemia , Transplante de Células-Tronco Hematopoéticas , Imunodeficiência Combinada Severa , Humanos , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/terapia , Adenosina Desaminase/genética , Terapia Genética/métodosRESUMO
Impairments of vision and hearing are highly prevalent conditions limiting the quality of life and presenting a major socioeconomic burden. For a long time, retinal and cochlear disorders have remained intractable for causal therapies, with sensory rehabilitation limited to glasses, hearing aids, and electrical cochlear or retinal implants. Recently, the application of gene therapy and optogenetics to eye and ear has generated hope for a fundamental improvement of vision and hearing restoration. To date, one gene therapy for the restoration of vision has been approved, and ongoing clinical trials will broaden its application including gene replacement, genome editing, and regenerative approaches. Moreover, optogenetics, i.e., controlling the activity of cells by light, offers a more general alternative strategy. Over little more than a decade, optogenetic approaches have been developed and applied to better understand the function of biological systems, while protein engineers have identified and designed new opsin variants with desired physiological features. Considering potential clinical applications of optogenetics, the spotlight is on the sensory systems, particularly the eye and ear. Multiple efforts have been undertaken to restore lost or hampered function in the eye and ear. Optogenetic stimulation promises to overcome fundamental shortcomings of electrical stimulation, namely, poor spatial resolution and cellular specificity, and accordingly to deliver more detailed sensory information. This review aims to provide a comprehensive reference on current gene therapeutic and optogenetic research relevant to the restoration of hearing and vision. We will introduce gene-therapeutic approaches and discuss the biotechnological and optoelectronic aspects of optogenetic hearing and vision restoration.
Assuntos
Perda Auditiva/terapia , Transtornos da Visão/terapia , Humanos , Optogenética , Próteses VisuaisRESUMO
The dark genome, the nonprotein-coding part of the genome, is replete with long noncoding RNAs (lncRNAs). These functionally versatile transcripts, with specific temporal and spatial expression patterns, are critical gene regulators that play essential roles in health and disease. In recent years, FAAH-OUT was identified as the first lncRNA associated with an inherited human pain insensitivity disorder. Several other lncRNAs have also been studied for their contribution to chronic pain and genome-wide association studies are frequently identifying single nucleotide polymorphisms that map to lncRNAs. For a long time overlooked, lncRNAs are coming out of the dark and into the light as major players in human pain pathways and as potential targets for new RNA-based analgesic medicines.
Assuntos
Dor , RNA Longo não Codificante , RNA Longo não Codificante/genética , Humanos , Dor/genética , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único/genética , Animais , Regulação da Expressão Gênica/genéticaRESUMO
Allogeneic hematopoietic stem cell transplantation is an effective treatment to cure inborn errors of immunity. Remarkable progress has been achieved thanks to the development and optimization of effective combination of advanced conditioning regimens and use of immunoablative/suppressive agents preventing rejection as well as graft versus host disease. Despite these tremendous advances, autologous hematopoietic stem/progenitor cell therapy based on ex vivo gene addition exploiting integrating γ-retro- or lenti-viral vectors, has demonstrated to be an innovative and safe therapeutic strategy providing proof of correction without the complications of the allogeneic approach. The recent advent of targeted gene editing able to precisely correct genomic variants in an intended locus of the genome, by introducing deletions, insertions, nucleotide substitutions or introducing a corrective cassette, is emerging in the clinical setting, further extending the therapeutic armamentarium and offering a cure to inherited immune defects not approachable by conventional gene addition. In this review, we will analyze the current state-of-the art of conventional gene therapy and innovative protocols of genome editing in various primary immunodeficiencies, describing preclinical models and clinical data obtained from different trials, highlighting potential advantages and limits of gene correction.
Assuntos
Edição de Genes , Transplante de Células-Tronco Hematopoéticas , Humanos , Edição de Genes/métodos , Terapia Genética/métodos , Vetores Genéticos/genéticaRESUMO
A number of endogenous genes in the human genome encode retroviral gag-like proteins, which were domesticated from ancient retroelements. The paraneoplastic Ma antigen (PNMA) family members encode a gag-like capsid domain, but their ability to assemble as capsids and traffic between cells remains mostly uncharacterized. Here, we systematically investigate human PNMA proteins and find that a number of PNMAs are secreted by human cells. We determine that PNMA2 forms icosahedral capsids efficiently but does not naturally encapsidate nucleic acids. We resolve the cryoelectron microscopy (cryo-EM) structure of PNMA2 and leverage the structure to design engineered PNMA2 (ePNMA2) particles with RNA packaging abilities. Recombinantly purified ePNMA2 proteins package mRNA molecules into icosahedral capsids and can function as delivery vehicles in mammalian cell lines, demonstrating the potential for engineered endogenous capsids as a nucleic acid therapy delivery modality.
Assuntos
Antígenos de Neoplasias , Capsídeo , Proteínas do Tecido Nervoso , Animais , Humanos , RNA Mensageiro/genética , Microscopia Crioeletrônica , MamíferosRESUMO
Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.
Assuntos
Sistemas CRISPR-Cas , Núcleo Celular , Edição de Genes , Terapia Genética , Músculo Esquelético , Distrofia Muscular de Duchenne , Edição de Genes/métodos , Animais , Camundongos , Músculo Esquelético/metabolismo , Núcleo Celular/metabolismo , Terapia Genética/métodos , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/genética , Humanos , Sinais de Localização Nuclear/genética , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Modelos Animais de Doenças , Mioblastos/metabolismoRESUMO
The complement alternative pathway (AP) is implicated in numerous diseases affecting many organs, ranging from the rare hematological disease paroxysmal nocturnal hemoglobinuria (PNH), to the common blinding disease age-related macular degeneration (AMD). Critically, the AP amplifies any activating trigger driving a downstream inflammatory response; thus, components of the pathway have become targets for drugs of varying modality. Recent validation from clinical trials using drug modalities such as inhibitory antibodies has paved the path for gene targeting of the AP or downstream effectors. Gene targeting in the complement field currently focuses on supplementation or suppression of complement regulators in AMD and PNH, largely because the eye and liver are highly amenable to drug delivery through local (eye) or systemic (liver) routes. Targeting the liver could facilitate treatment of numerous diseases as this organ generates most of the systemic complement pool. This review explains key concepts of RNA and DNA targeting and discusses assets in clinical development for the treatment of diseases driven by the alternative pathway, including the RNA-targeting therapeutics ALN-CC5, ARO-C3, and IONIS-FB-LRX, and the gene therapies GT005 and HMR59. These therapies are but the spearhead of potential drug candidates that might revolutionize the field in coming years.