For several decades, exploring and treating genetic diseases has posed a great challenge for the scientific and medical community. Improvements in our understanding of the molecular basis of conditions have led to the discovery of novel approaches to treat these disorders with nucleic acid-based drugs. The ground-breaking discovery in the 1970s that genes can be introduced into eukaryotic cells by transduction with viral vectors (Sambrook et al., 1968) paved the way for the first clinical gene therapy trials. Since then, gene therapy has become a viable strategy for treating genomic and/or transcriptomic diseases by replacing, blocking, or editing individual genes or gene products (i.e., proteins) inside the cells (Bulcha et al., 2021). However, the first gene therapy clinical trial in 1990 used a viral vector to treat adenosine deaminase (ADA) deficiency, raising serious concerns regarding the safety and efficacy of viral vectors for gene therapy. Therefore, attention was garnered toward using safer non-viral vectors for gene delivery.

Compared to viral vectors, non-viral vectors are, in most cases, easier to manufacture, and they display lower host immunogenicity (Xu et al., 2018) and carcinogenesis (Babaei et al., 2020). Besides these advantages, these vector systems can be loaded with more genetic material and delivered to the target cells with techniques that are easy to adapt to any biological system (Zu and Gao, 2021). Non-viral vectors show great diversity, yet they can broadly be classified into (i) inorganic particles (Luther et al., 2020), (ii) polymer-based delivery systems (Rideau et al., 2018), (iii) protein-based delivery systems (Hong et al., 2020), and (iv) lipid-based delivery systems (LDS) (Hou et al., 2021). Although the reduced delivery efficiency, compared to the efficiency of viral vectors, represents a major drawback for non-viral vectors, lipid-based gene delivery systems are still one of the most promising technologies, especially for gene therapy-oriented clinical trials. Assessing and optimizing specific quality aspects before clinical use is crucial. This ensures effective cellular uptake and escape from endosomes, particularly for exogenous DNA entering the nucleus, which involves intricate and regulated biomolecular interactions. (Behzadi et al., 2017). In particular, designing lipid molecules for their manufacturing, selecting the genetic material payload, and encapsulation methods are crucial parameters in the design of these vector systems.

LDSs can be divided into two categories based on their vesicle-forming ability: (1) vesicular and (2) non-vesicular carriers. Vesicular aggregation offers advantages, as these vesicles can precisely mimic the cell membrane, resulting in easier interactions with cells (Mattern-Schain et al., 2019). They are supramolecular colloidal systems composed of one or several amphiphilic lipids, and conventional LDSs are composed of phospholipids and cholesterol (Chol). Closed membrane structures, called liposomes, are self-assembled when amphiphilic molecules with a hydrophilic headgroup and a hydrophobic tail are dispersed in an aqueous system (Bangham et al., 1965). Such a vector system can entrap hydrophilic drugs, e.g., genetic material, in the aqueous core, and hydrophobic drugs can be embedded into the membrane bilayer. Unlike vesicular counterparts, non-vesicular LDSs demonstrate great differences in chemical structure, lipid orientation and composition, and genetic cargo release (Hou et al., 2021). They are multi-component systems composed of an ionizable lipid, a phospholipid, and cholesterol. The ionizable lipid possesses a cationic nature and complexes with genetic cargo via electrostatic attraction, forming a core structure. In the meantime, helper lipids serve as an envelope to enclose the network of mRNA-containing core structures (Eygeris et al., 2020).

LDSs composed of lipids only as building blocks are considered conventional systems (Fig. 1), usually used for passive targeting of phagocytic immune cells. For more specific gene delivery applications, non-functionalized LDSs typically exhibit reduced efficiency in transient transgene expression, leading to less than optimal therapeutic responses. (Liu et al., 2020). Furthermore, they have a limitation in protecting genetic materials from degradation in the physiological fluids due to the robust action of phagocytes of the immune system, as well as endosomes inside the cells, which is a great challenge in gene delivery approaches (Guimarães et al., 2021). Therefore, LDSs are often modified chemically or by adding certain polymers. This extends their circulation time, improves targeting, safeguards against degradation, and enhances therapeutic effectiveness for better delivery success (Luiz et al., 2022). In addition, genetically defective cells (e.g., cancer cells) overexpress certain receptor proteins on their surfaces. Still, conventional delivery systems do not possess the functional therapeutic structure to target these receptors (Tie et al., 2020). Due to chemically active substituents or moieties present within these systems and their high affinity for several chemical groups on the cell surface, engineering with specific chemical ligands (e.g., aptamers, peptides, or antibodies) represents an important strategy to enhance the therapeutic efficacy of nucleic acid-based cargoes, and consequently, to increase their success rate in clinical trials.

This review aims to comprehensively delve into engineering strategies for functionalizing LDSs and highlight their therapeutic efficacy for delivering novel genetic cargoes for in vivo applications to provide a full snapshot of the translation from the bench to the clinics and market. Besides conventional lipid-based vesicular systems, lipid nanoparticles, despite their dissimilarity in structure, are also discussed to provide an overview of contemporary trends in gene therapy. Surface functionalization of delivery systems using antibodies for cancer therapy is a concern, but the potential use of these gene delivery systems for vaccination purposes is excluded from this review. We have also introduced strategies showing the co-administration of nucleic acids with chemotherapeutic drugs to explore their mutual effect, emphasizing the challenges these LDSs face when co-delivering these two distinct cargos with different natures. Although these delivery systems have been widely employed for gene delivery, their efficiency is hindered by several factors, as detailed in this review. Here, we outline existing challenges in the delivery and internalization processes and narrate recent advances in the functionalization of delivery systems to enhance their therapeutic efficacy and safety. Moreover, clinical trials use these vectors to expand their clinical use and address their principal safety concerns.