Gene therapies are powerful tools for the treatment of inflammatory, genetic, and cancer-related skin diseases.The skin barrier function and the low number of cells that get transfected are the main hurdles for cutaneous gene therapy and contribute to the fact that gene therapies for skin diseases are an underexplored area.Gene editing provides an approach to cure rare and severe genodermatoses-like epidermolysis bullosa. First studies demonstrate the potential and invaluable impact these treatments may have even if only a small percentage of the gene function can be restored.Recent advancements demonstrate the power of non-viral delivery systems for the delivery of gene therapeutics to the skin. They may prove superior to viral vectors, the current gold standard, because their use is not limited by packaging size, serious safety concerns, or manufacturing issues. Gene therapies are powerful tools to prevent, treat, and cure human diseases. The application of gene therapies for skin diseases received little attention so far, despite the easy accessibility of skin and the urgent medical need. A major obstacle is the unique barrier properties of human skin, which significantly limits the absorption of biomacromolecules, and thus hampers the efficient delivery of nucleic acid payloads. In this review, we discuss current approaches, successes, and failures of cutaneous gene therapy and provide guidance toward the development of next-generation concepts. We specifically allude to the delivery strategies as the major obstacle that prevents the full potential of gene therapies – not only for skin disorders but also for almost any other human disease. Gene therapies are powerful tools to prevent, treat, and cure human diseases. The application of gene therapies for skin diseases received little attention so far, despite the easy accessibility of skin and the urgent medical need. A major obstacle is the unique barrier properties of human skin, which significantly limits the absorption of biomacromolecules, and thus hampers the efficient delivery of nucleic acid payloads. In this review, we discuss current approaches, successes, and failures of cutaneous gene therapy and provide guidance toward the development of next-generation concepts. We specifically allude to the delivery strategies as the major obstacle that prevents the full potential of gene therapies – not only for skin disorders but also for almost any other human disease. Gene therapies, including RNA-based approaches, gene augmentation, and gene editing (Table 1), are powerful tools to prevent, treat, and cure a multitude of human diseases [1.Doudna J.A. The promise and challenge of therapeutic genome editing.Nature. 2020; 578: 229-236Crossref PubMed Scopus (559) Google Scholar]. The first gene therapy, Glybera (see Glossary), obtained regulatory approval in 2012 and at least eight more followed since then. Modern cutaneous gene therapy was pioneered by Paul Khavari and his group, who published first studies on the delivery of the transglutaminase 1 gene into congenital ichthyosis patient cells using retroviruses back in 1996 [2.Choate K.A. et al.Transglutaminase 1 delivery to lamellar ichthyosis keratinocytes.Hum. Gene Ther. 1996; 7: 2247-2253Crossref PubMed Scopus (74) Google Scholar]. Since then, incredibly fast advancements, especially in the field of CRISPR-based gene editing, have propelled the potential applications of gene therapies. Hence, the drug development pipelines and clinical trials are full of gene therapy-based approaches that provide a new lever for the treatment of previously untreatable conditions [3.Farboud B. et al.Enhanced genome editing with Cas9 ribonucleoprotein in diverse cells and organisms.J. Vis. Exp. 2018; 57350Google Scholar]. Interestingly, the application of gene therapies for skin-related diseases has received comparably little attention so far, despite the easy accessibility of human skin and the urgent medical need [4.Bilousova G. Gene therapy for skin fragility diseases: the new generation.J. Invest. Dermatol. 2019; 139: 1634-1637Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar].Table 1Overview of the Current Gene Therapy ToolsToolsFeaturesAdvantagesChallengesRNA-based therapiesa) Messenger RNA (mRNA)• Induce the production of desired protein to restore normal function Promising for 'undruggable' targets Easy and rapid chemical synthesis Cost effective and stable shelf life Allows personalized medicineLow immunogenicity. Poor pharmacokinetic properties Variable efficacy in suppression of target protein Off-target effectsb) Silencing RNA• Short double-stranded RNA fragments• Triggers mRNA degradation or blocks its transcriptionsc) AON• Short sequences of modified DNA or RNA• Inhibit mRNA translation into proteinsGene augmentationa) Plasmid DNA• Wild-type copy of mutated gene• Transient gene knock-in or DNA-directed RNA interference (gene knockdown) Regulated gene expression Easy to design and cost effective Flexible for personalized medicine Low immunogenicity Translocation into the cell nucleus is required Readministration is required for long-lasting effectsb) Minicircle DNAc) Mini-string DNAGene repair and editingInsertion, deletion, or replacement of genesa) ZFN and b) TALEN proteins detect target DNA sequences.• Programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domainBroad range of gene editingTime-consuming and labor-intensive de novo protein engineeringc) RNA-guided nuclease technology (CRISPR/Cas9)• A target-specific guide RNA (gRNA) complexes with the Cas9 nuclease protein• Induction of double-strand breaks (DSBs) at target site•DSBs are repaired either by nonhomologous end joining or homology-directed repair Cas nucleases can be delivered as DNA, mRNA, or RNP, which refers to preassembled Cas9 protein–gRNA complex Fast, cheap, simple RNP: higher editing rates and less off-target effects than plasmid-based Cas expression Transfection is challenging Risk of off-target effects uncleara) Base editing•An impaired Cas9–sgRNA combined with deaminase (a catalytic enzyme which permanently alters the chemical sequence of single base). Conversion of single bases or base pairs possible No DSBs Correction of single-nucleotide polymorphism Transfection is challenging Risk of off-target effects remains unclear Open table in a new tab An impaired skin barrier may pose a serious threat to health, and skin diseases significantly affect physical and psychological well-being. In fact, they belong to the most frequent diseases of humans causing a significant economic burden worldwide [5.Laughter M.R. et al.The burden of skin and subcutaneous diseases in the United States from 1990 to 2017.JAMA Dermatol. 2020; (Published online June 10, 2020. https://doi.org/10.1001/jamadermatol.2020.1573)Crossref PubMed Scopus (34) Google Scholar]. In principle, all types of skin diseases are candidates for gene therapy, ranging from inflammatory diseases over skin cancer to genodermatoses. Genodermatoses are a diverse group of rare, often severe skin diseases that result from a variety of single mutations in 500 or more genes. One of the most common genodermatoses is epidermolysis bullosa (EB), a rare genetic condition that results in blistering of the skin and mucous membranes with severity ranging from mild to fatal [6.Pope E. Epidermolysis bullosa: a 2020 perspective.Br. J. Dermatol. 2020; (Published online May 10, 2020. https://doi.org/10.1111/bjd.19125)Crossref Scopus (5) Google Scholar]. While EB persists into adulthood, it is especially significant in neonates who suffer from higher mortality rates in some instances because of dehydration and infection [6.Pope E. Epidermolysis bullosa: a 2020 perspective.Br. J. Dermatol. 2020; (Published online May 10, 2020. https://doi.org/10.1111/bjd.19125)Crossref Scopus (5) Google Scholar,7.Oji V. et al.Revised nomenclature and classification of inherited ichthyoses: results of the First Ichthyosis Consensus Conference in Soreze 2009.J. Am. Acad. Dermatol. 2010; 63: 607-641Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar]. Further examples for genodermatoses are congenital ichthyosis and the Netherton syndrome. So far, genodermatoses cannot be cured, and current treatment options purely rely on the relief of symptoms. A potentially curative strategy is to repair the disease-causing mutations within the host genome using gene editing [1.Doudna J.A. The promise and challenge of therapeutic genome editing.Nature. 2020; 578: 229-236Crossref PubMed Scopus (559) Google Scholar,8.Arney K. Change the genes to fix the skin.Nature. 2018; 564: S14-S15Crossref PubMed Scopus (2) Google Scholar]. This, together with the high medical need, makes genodermatoses the prime candidate for cutaneous gene therapy. Overall, however, the absorption of biomacromolecules, such as gene therapeutics into the skin, is highly restricted due to its unique composition and structural organization (Figure 1; Box 1). In fact, human skin only allows efficient absorption of small [molecular weight (MW) ≤ 800 Da] and moderately lipophilic (logP 1–3) molecules [9.Lundborg M. et al.Human skin barrier structure and function analyzed by cryo-EM and molecular dynamics simulation.J. Struct. Biol. 2018; 203: 149-161Crossref PubMed Scopus (57) Google Scholar,10.Kabashima K. et al.The immunological anatomy of the skin.Nat. Rev. Immunol. 2019; 19: 19-30Crossref PubMed Scopus (353) Google Scholar], which makes it an exceptionally challenging target for gene delivery.Box 1Structure of Human SkinHuman skin exerts several vital functions as it protects the human body from excessive transepidermal water loss and prevents the entry of xenobiotics and microbes. It is characterized by a highly sophisticated structural organization and is composed of three main layers: the epidermis, the dermis, and the hypodermis. Epidermis and dermis are connected by the basement membrane, epidermal–dermal junction, which also anchors the epidermis and dermis through proteins such as collagen and integrins, providing resistance against external shear stress.The epidermis encompasses three main cell populations: keratinocytes, which make up 99% of the cells, melanocytes, and highly specialized immune cells, the Langerhans cells. Keratinocytes derive from the skin stem cells, which are located in the stratum basale, then undergo continuous maturation and differentiation, forming three different epidermal layers: the stratum spinosum, stratum granulosum, and SC (Figure 1) [9.Lundborg M. et al.Human skin barrier structure and function analyzed by cryo-EM and molecular dynamics simulation.J. Struct. Biol. 2018; 203: 149-161Crossref PubMed Scopus (57) Google Scholar,10.Kabashima K. et al.The immunological anatomy of the skin.Nat. Rev. Immunol. 2019; 19: 19-30Crossref PubMed Scopus (353) Google Scholar]. Except for the SC, all skin layers are metabolically active. The outermost layer of the human skin, the SC, consists of corneocytes – terminally differentiated keratinocytes which are interconnected by keratin filaments and are enclosed within an insoluble amalgam of crosslinked skin proteins. The corneocytes are embedded in a highly lipophilic skin lipid matrix. The lipid matrix is a mixture of ceramides, cholesterol, and fatty acids, organized in an orthorhombic lattice packing that determines the unique barrier properties of human skin.The dermis is an elastic connective tissue which is highly vascularized and, thus, supports the epidermis as well as the skin appendages, such as hair follicles, sweat, and sebaceous glands. Fibroblasts, the primary cell type of the dermis, produce and secrete structural proteins such as collagen fibers, elastin, and proteoglycans that together form the extracellular matrix, in which further immune cells such as macrophages, mast cells, and dendritic cells are embedded.The hypodermis is rich in adipose tissue, collagen, and elastic fibers and, thus, acts as a cushion and protects the body against temperature variations. This loose connective tissue further aids the support of nerves and blood vessels in the skin. Human skin exerts several vital functions as it protects the human body from excessive transepidermal water loss and prevents the entry of xenobiotics and microbes. It is characterized by a highly sophisticated structural organization and is composed of three main layers: the epidermis, the dermis, and the hypodermis. Epidermis and dermis are connected by the basement membrane, epidermal–dermal junction, which also anchors the epidermis and dermis through proteins such as collagen and integrins, providing resistance against external shear stress. The epidermis encompasses three main cell populations: keratinocytes, which make up 99% of the cells, melanocytes, and highly specialized immune cells, the Langerhans cells. Keratinocytes derive from the skin stem cells, which are located in the stratum basale, then undergo continuous maturation and differentiation, forming three different epidermal layers: the stratum spinosum, stratum granulosum, and SC (Figure 1) [9.Lundborg M. et al.Human skin barrier structure and function analyzed by cryo-EM and molecular dynamics simulation.J. Struct. Biol. 2018; 203: 149-161Crossref PubMed Scopus (57) Google Scholar,10.Kabashima K. et al.The immunological anatomy of the skin.Nat. Rev. Immunol. 2019; 19: 19-30Crossref PubMed Scopus (353) Google Scholar]. Except for the SC, all skin layers are metabolically active. The outermost layer of the human skin, the SC, consists of corneocytes – terminally differentiated keratinocytes which are interconnected by keratin filaments and are enclosed within an insoluble amalgam of crosslinked skin proteins. The corneocytes are embedded in a highly lipophilic skin lipid matrix. The lipid matrix is a mixture of ceramides, cholesterol, and fatty acids, organized in an orthorhombic lattice packing that determines the unique barrier properties of human skin. The dermis is an elastic connective tissue which is highly vascularized and, thus, supports the epidermis as well as the skin appendages, such as hair follicles, sweat, and sebaceous glands. Fibroblasts, the primary cell type of the dermis, produce and secrete structural proteins such as collagen fibers, elastin, and proteoglycans that together form the extracellular matrix, in which further immune cells such as macrophages, mast cells, and dendritic cells are embedded. The hypodermis is rich in adipose tissue, collagen, and elastic fibers and, thus, acts as a cushion and protects the body against temperature variations. This loose connective tissue further aids the support of nerves and blood vessels in the skin. Hence, the focus of this review is to provide insight into the strengths and limitations of skin-relevant gene delivery strategies. In fact, efficient, targeted, and safe gene delivery is the major obstacle that currently hampers the translation of gene therapies from bench-to-bedside – not only for skin disorders but also for almost any other human disease [11.van Haasteren J. et al.The delivery challenge: fulfilling the promise of therapeutic genome editing.Nat. Biotechnol. 2020; 38: 845-855Crossref PubMed Scopus (148) Google Scholar]. Further, we discuss the successes and failures of intradermal gene therapy and provide guidance toward the development of next-generation concepts. Viral vectors currently are the most effective carriers for gene delivery due to their innate capability to infect both dividing and quiescent cells. Currently, 70% of ongoing gene therapy clinical trials are viral vector based [12.Picanço-Castro V. et al.Emerging patent landscape for non-viral vectors used for gene therapy.Nat. Biotechnol. 2020; 38: 151-157Crossref PubMed Scopus (54) Google Scholar]. Nonintegrating viral vectors, such as adenoviral vectors and adeno-associated viral vectors (AAVs), offer the advantage of inherent infection ability without triggering potential problems due to insertional mutagenesis [13.Kimura T. et al.Production of adeno-associated virus vectors for in vitro and in vivo applications.Sci. Rep. 2019; 9: 13601Crossref PubMed Scopus (77) Google Scholar]. AAVs seem to be less immunogenic than other viruses, but the risk of pre-existing immunity limits their transfection efficacy and prevents re-dosing regimens [14.Valdmanis P.N. et al.rAAV-mediated tumorigenesis: still unresolved after an AAV assault.Mol. Ther. 2012; 20: 2014-2017Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar]. By contrast, lentiviral vectors, which belong to the retroviruses, maintain stable long-term transgene expression but are capable of genome integration, which increases the risk for insertional mutagenesis [15.Palfi S. et al.Long-term follow-up of a phase I/II study of ProSavin, a lentiviral vector gene therapy for Parkinson's disease.Hum. Gene Ther. Clin. Dev. 2018; 29: 148-155Crossref PubMed Scopus (92) Google Scholar]. Despite their efficacy, the use of viral vectors is accompanied by considerable challenges, the biggest of which is the limited cargo size. AAVs (20 nm), for example, can only encapsulate approximately 4.7 kb of genetic cargo. Gene-editing tools such as CRISPR require a much larger and/or additional carriers for efficient delivery. To overcome the size caveat, the use of dual vectors has been proposed: one for the Cas9 and the other for the single guide RNA (sgRNA) [16.Yang Y. et al.A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice.Nat. Biotechnol. 2016; 34: 334-338Crossref PubMed Scopus (424) Google Scholar]. However, the success of this approach relies on the simultaneous intracellular delivery of sgRNA and Cas9. Further, curative approaches for recessive genodermatoses require the simultaneous delivery of donor templates, which cannot be achieved using viral vectors. Recent advancements of the CRISPR-Cas technology, such as prime editing, enable more precise cutting and higher editing efficacies, but viral vectors cannot serve as delivery tools as the reverse transcriptase (7 kb) cannot be encapsulated [17.Anzalone A.V. et al.Search-and-replace genome editing without double-strand breaks or donor DNA.Nature. 2019; 576: 149-157Crossref PubMed Scopus (2415) Google Scholar]. Although lentiviral vectors (80–100 nm) can encapsulate larger genomic materials (approximately 10 kb) [18.Koike-Yusa H. et al.Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.Nat. Biotechnol. 2014; 32: 267-273Crossref PubMed Scopus (803) Google Scholar,19.Kabadi A.M. et al.Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.Nucleic Acids Res. 2014; 42: e147Crossref PubMed Scopus (257) Google Scholar], their safety remains controversially discussed [15.Palfi S. et al.Long-term follow-up of a phase I/II study of ProSavin, a lentiviral vector gene therapy for Parkinson's disease.Hum. Gene Ther. Clin. Dev. 2018; 29: 148-155Crossref PubMed Scopus (92) Google Scholar]. In addition to packaging constraints, the broad tissue-targeting ability of viral vectors can be problematic as the long-term expression of gene-editing molecules may result in off-target effects and immunogenicity [20.Charlesworth C.T. et al.Identification of preexisting adaptive immunity to Cas9 proteins in humans.Nat. Med. 2019; 25: 249-254Crossref PubMed Scopus (578) Google Scholar]. Finally, the design of viral vectors requires complex manufacturing processes at the highest standards, which raises cost-related problems [21.Senior M. After Glybera's withdrawal, what's next for gene therapy?.Nat. Biotechnol. 2017; 35: 491-492Crossref PubMed Scopus (73) Google Scholar]. Today, lipid-based nanoparticles (LNPs) are the most advanced, non-viral gene delivery systems. In fact, the approval of ONPATTRO in 2018, the first-ever siRNA-based drug, has paved the way for a new class of gene therapies. LNPs have become a clinically validated platform technology to deliver genetic material of nearly any size, for example, ≤20-kbp DNA vectors have been successfully delivered in a preclinical setting [22.Fink T.L. et al.Plasmid size up to 20 kbp does not limit effective in vivo lung gene transfer using compacted DNA nanoparticles.Gene Ther. 2006; 13: 1048-1051Crossref PubMed Scopus (123) Google Scholar], and about 11.5-kb self-amplifying RNAs encoding for SARS-CoV-2 (severe acute respiratory syndrome-coronavirus 2) proteins are currently being tested in the clinic as COVID-19 (coronavirus disease 2019) vaccines [23.McKay P.F. et al.Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice.Nat. Commun. 2020; 113523Crossref PubMed Scopus (311) Google Scholar]. This revolutionary discovery offers the potential to treat human skin disorders by silencing pathogenic genes, expressing therapeutic proteins, or correcting genetic defects [24.Akinc A. et al.The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs.Nat. Nanotechnol. 2019; 14: 1084-1087Crossref PubMed Scopus (807) Google Scholar]. Rapid mixing and microfluidic procedures enable efficient large-scale manufacturing crucial for entering into routine clinical practice. Until recently, however, only a few studies explored the use of LNPs for treating skin disorders. Historically, cationic lipids [such as 1,2-dioleoyl-3-trimethylammonium propane, (DOTAP)] in combination with helper lipids (e.g., phospholipids or cholesterol) have been used to complex, protect, and deliver nucleic acids [25.Buck J. et al.Lipid-based DNA therapeutics: hallmarks of non-viral gene delivery.ACS Nano. 2019; 13: 3754-3782Crossref PubMed Scopus (230) Google Scholar]. These lipoplexes and related transfection reagents such as Lipofectamine are powerful agents for introducing gene constructs into cells in vitro. Although promising, these systems have little clinical utility due to carrier-related toxicity attributed to the permanent cationic charge and immune activation. In addition, certain cationic lipids, including DOTAP, have even shown inhibitory effects for efficient cutaneous gene transfer [26.Yu W.H. et al.Topical gene delivery to murine skin.J. Invest. Dermatol. 1999; 112: 370-375Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar,27.Blakney A.K. et al.The skin you are in: design-of-experiments optimization of lipid nanoparticle self-amplifying RNA formulations in human skin explants.ACS Nano. 2019; 13: 5920-5930Crossref PubMed Scopus (42) Google Scholar]. A key advancement of LNP technology was the identification of ionizable cationic lipids such as DLin-MC3-DMA [28.Jayaraman M. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (854) Google Scholar,29.Semple S.C. et al.Rational design of cationic lipids for siRNA delivery.Nat. Biotechnol. 2010; 28: 172-176Crossref PubMed Scopus (1326) Google Scholar]. This class of lipids possesses an acid dissociation constant (pKa) of approximately 6.5. This ensures that the lipid is neutral under physiological conditions [28.Jayaraman M. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (854) Google Scholar], and positively charged at acidic pH to enable efficient entrapment of nucleic acids. This discovery significantly reduced carrier-related side effects and thus improved the therapeutic index by several orders of magnitude. Upon LNP internalization, the low endosomal pH allows for protonation of the ionizable lipid, resulting in destabilization of the endosome via interaction with negatively charged endosomal lipids, and finally cytoplasmic release of the genetic payload (Box 2) [30.Kulkarni J.A. et al.Lipid nanoparticle technology for clinical translation of siRNA therapeutics.Acc. Chem. Res. 2019; 52: 2435-2444Crossref PubMed Scopus (266) Google Scholar]. Conclusively, the ionizable lipid has a threefold function: efficient interaction and entrapment with nucleic acid drugs, reduction of particle toxicity, and facilitation of endosomal escape [28.Jayaraman M. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (854) Google Scholar].Box 2Extra- and Intracellular Barriers for Non-viral Delivery SystemsNon-viral gene delivery systems such as lipid nanoparticles (LNPs) or polymeric nanoparticles face several extracellular and intracellular barriers that must be overcome before generating any therapeutic effect. Consequently, non-viral delivery systems are designed to prevent the degradation of genetic material through endonucleases, escape immune detection, and reduce nonspecific interactions [25.Buck J. et al.Lipid-based DNA therapeutics: hallmarks of non-viral gene delivery.ACS Nano. 2019; 13: 3754-3782Crossref PubMed Scopus (230) Google Scholar]. In addition, the delivery systems need to reach the target tissue, enter the cells, escape the endosomes, and unpack the genetic material. For RNA-based therapies, delivery into the cytosol is sufficient, whereas for DNA vectors and gene-editing tools, the nucleus needs to be reached. Cellular uptake mechanisms of non-viral delivery systems include adsorptive or receptor-mediated endocytosis. The latter is based on specific interactions of endogenous (i.e., serum proteins covering nanoparticles) or exogenous (i.e., chemically conjugated moieties) targeting ligands on the nanoparticle with cell surface receptors. Once internalized, the non-viral delivery systems become hostile within the endocytic vesicles that rapidly acidify to pH 5–6. In the case of polymer-based gene delivery systems, proton-accepting amines prevent the acidification, leading to osmotic vesicle swelling and finally endosomal membrane rupture (proton sponge hypothesis) [92.Lai W.F. Wong W.T. Design of polymeric gene carriers for effective intracellular delivery.Trends Biotechnol. 2018; 36: 713-728Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar]. By contrast, the protonation of lipids within the acidic endosomes triggers an association with anionic endosomal lipids. The formation of membrane disruptive non-lamellar structures facilitates the subsequent endosomal escape (shape hypothesis) [93.Kulkarni J.A. et al.On the role of helper lipids in lipid nanoparticle formulations of siRNA.Nanoscale. 2019; 11: 21733-21739Crossref PubMed Google Scholar].Overall, the performance of non-viral delivery systems can be enhanced by both chemical and physical engineering strategies. Size, zeta potential, and geometric optimization are mandatory, and chemical engineering, such as the conjugation of targeting ligands and cell-penetrating peptides or the generation of stimuli-responsive nanoparticles, may facilitate the cellular entry and endosomal escape [30.Kulkarni J.A. et al.Lipid nanoparticle technology for clinical translation of siRNA therapeutics.Acc. Chem. Res. 2019; 52: 2435-2444Crossref PubMed Scopus (266) Google Scholar]. Non-viral gene delivery systems such as lipid nanoparticles (LNPs) or polymeric nanoparticles face several extracellular and intracellular barriers that must be overcome before generating any therapeutic effect. Consequently, non-viral delivery systems are designed to prevent the degradation of genetic material through endonucleases, escape immune detection, and reduce nonspecific interactions [25.Buck J. et al.Lipid-based DNA therapeutics: hallmarks of non-viral gene delivery.ACS Nano. 2019; 13: 3754-3782Crossref PubMed Scopus (230) Google Scholar]. In addition, the delivery systems need to reach the target tissue, enter the cells, escape the endosomes, and unpack the genetic material. For RNA-based therapies, delivery into the cytosol is sufficient, whereas for DNA vectors and gene-editing tools, the nucleus needs to be reached. Cellular uptake mechanisms of non-viral delivery systems include adsorptive or receptor-mediated endocytosis. The latter is based on specific interactions of endogenous (i.e., serum proteins covering nanoparticles) or exogenous (i.e., chemically conjugated moieties) targeting ligands on the nanoparticle with cell surface receptors. Once internalized, the non-viral delivery systems become hostile within the endocytic vesicles that rapidly acidify to pH 5–6. In the case of polymer-based gene delivery systems, proton-accepting amines prevent the acidification, leading to osmotic vesicle swelling and finally endosomal membrane rupture (proton sponge hypothesis) [92.Lai W.F. Wong W.T. Design of polymeric gene carriers for effective intracellular delivery.Trends Biotechnol. 2018; 36: 713-728Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar]. By contrast, the protonation of lipids within the acidic endosomes triggers an association with anionic endosomal lipids. The formation of membrane disruptive non-lamellar structures facilitates the subsequent endosomal escape (shape hypothesis) [93.Kulkarni J.A. et al.On the role of helper lipids in lipid nanoparticle formulations of siRNA.Nanoscale. 2019; 11: 21733-21739Crossref PubMed Google Scholar]. Overall, the performance of non-viral delivery systems can be enhanced by both chemical and physical engineering strategies. Size, zeta potential, and geometric optimization are mandatory, and chemical engineering, such as the conjugation of targeting ligands and cell-penetrating peptides or the generation of stimuli-responsive nanoparticles, may facilitate the cellular entry and endosomal escape [30.Kulkarni J.A. et al.Lipid nanoparticle technology for clinical translation of siRNA ther