Nano Approaches to Nucleic Acid Delivery: Barriers, Solutions, and Current Landscape

ABSTRACT

Nucleic acid (NA) therapy holds tremendous potential for treating a wide range of genetic diseases by the delivery of therapeutic genes into target cells. However, significant challenges exist in safely and effectively delivering these genes to their intended locations. Viral vectors, though efficient, pose risks such as immunogenicity and mutagenesis. This has resulted in growing interest in non‐viral, nanoparticle‐based NA delivery systems. This review article describes various physiological barriers to NA delivery and explores nanoparticle‐based NA delivery systems, including bioengineered nanoparticles, peptides, lipid nanoparticles, and polymeric nanoparticles, highlighting their unique features to overcome in vivo barriers for NA delivery. While these nanoparticle‐based NA delivery systems offer a promising alternative to viral vectors, challenges related to cytotoxicity, reproducible synthesis, and cost need to be addressed. The current clinical landscape of NA delivery is also discussed, emphasizing the need for safer, scalable, and cost‐effective solutions. Nanoparticles represent a promising future in NA therapy, with the possibility of developing clinically relevant, non‐toxic, stable, and non‐immunogenic delivery vehicles, paving the way for broader therapeutic applications and improved clinical outcomes.

Nano approaches for breaking barriers to nucleic acid delivery.

1. Introduction

Nucleic acid (NA) therapy has revolutionized the field of medicine by providing tailored treatment options, surpassing what conventional approaches can achieve. NA therapy possesses great potential for the treatment of genetic mutations that disrupt regular cellular mechanisms, causing cell proliferation, metastasis, immunogenicity, and angiogenesis, ending up in complex diseases. The idea of NA therapy was conceptualized by the physician Theodore Friedmann and biochemist Richard Roblin in 1972 (Friedmann and Roblin 1972). NA therapy, as a concept, rectifies the proximate cause of the disease at the cellular level by replacing or restoring the defective genes with healthy variants to prevent or treat a particular disease. This strategy aims to either inactivate the malfunctioning genes by knocking out the mutated genes or delivering new gene codes to counteract existing abnormalities (Massadeh et al. 2016). While traditional treatments show positive effects, most malignancies remain resistant due to a limited understanding of the molecular mechanisms involved in their pathology. NA therapy, however, can offer curative long‐term solutions for a wide range of diseases, including but not limited to cancer, autoimmune diseases, neurodegenerative diseases, cardiovascular diseases, diabetes, cystic fibrosis, AIDS, and hemophilia (Beitelshees et al. 2017). The recent approval of various nucleic acid (NA) therapeutics by the United States Food and Drug Administration (US FDA), including mRNA vaccines for COVID‐19, has demonstrated the clinical potential of NA therapy for unmet medical needs with undruggable targets. As a result, NA therapy has rapidly boomed in several research areas in recent decades by successfully translating experimental trials into viable clinical settings (Figure 1) and leveraging a new pathway for effective therapies for unmet medical conditions.

FIGURE 1.

Selected milestones in the history of nucleic acid‐based therapies including recent approvals by the FDA. ASO: Anti‐sense oligonucleotide; CMV: Cytomegalovirus; CRISPR: Clustered regularly interspaced short palindromic repeats; DMD: Duchenne muscular dystrophy; GalNAc: N‐acetyl galactosamine; LNP: Lipid nanoparticles; rAAV: Recombinant adeno‐associated virus; SMA: Spinal muscular atrophy; TALEN: Transcription activator‐like effector nucleases; ZFN: Zinc finger nucleases. Created in BioRender.com .

Despite the high potential in therapeutic benefits, NA therapy faces considerable challenges that hinder its widespread medical applications. The primary obstacle of NA therapy is ineffective delivery to target sites. The NA delivery should be accurate and precise to minimize unintended genetic off‐target effects. It is essential that gene codes reach the nucleus, where gene transfer occurs. Due to the large size and negative charge, genomic materials struggle to penetrate the targeted membrane barrier, and locating the nucleus can be difficult (Roma‐Rodrigues et al. 2020). Even if they do reach the nucleus, their stability can be compromised by the dynamic cellular environment and the body's defense mechanism. For instance, nuclease‐mediated degradation inactivates the functional capacity, significantly impacting the sustained therapeutic effects and necessitating repeated interventions to maintain efficacy. Safety is another critical concern because the most common carriers for gene therapies are viral vectors; handling viral genomes can be challenging due to toxicity and the potential to trigger uncontrollable immune responses in the host genome, leading to insertional mutagenesis and raising the risk of oncogenesis (Massadeh et al. 2016). Additionally, the financial burden associated with developing, formulating, and manufacturing gene therapies, as well as administering them, not only restricts their clinical availability but also underscores the disparity between scientific breakthroughs and their practical translation in healthcare.

With the rapid emergence of nanocarriers in medical applications and their potential to encounter the existing barriers in NA therapy, nano‐based NA delivery systems are becoming a focal point of research, offering novel solutions. The versatility and tunable properties of nanocarriers make them well‐suited for these NA therapy applications with ideal pharmacokinetic abilities. Notably, many nanocarriers are already used extensively in targeted drug delivery, allowing gene therapies to be more precisely directed toward diseased cells (Liaw et al. 2021; Pitha et al. 2023; Porterfield et al. 2023; Sharma, Liaw, et al. 2020; Sharma et al. 2021). In extracellular conditions, nanocarriers improve circulation time and evade immune recognition while also addressing solubility issues. Intracellularly, genomic materials can be engineered to encapsulate within nanocarriers (liposomes, dendrimers, and inorganic nanoparticles), protecting them from enzymatic degradation (Massadeh et al. 2016). Dendrimers, a class of hyperbranched polymers, offer high surface multivalency, allowing for the conjugation of multiple genomic materials and therapeutic agents (Sharma et al. 2014; Sharma, Kambhampati, et al. 2020; Tarach and Janaszewska 2021). Additionally, inorganic nanoparticles possess unique optical and magnetic properties that aid in imaging, enabling the monitoring of gene delivery and distribution within the body (Lin et al. 2021).

The delivery and therapeutic efficacy of NAs, such as DNA, mRNA, siRNA, and ASOs, present various challenges. These challenges largely stem from the need for nuclear entry and the susceptibility of these NAs to nuclease degradation (Xu and Anchordoquy 2011). For DNA, nuclear entry is essential for transcription into mRNA and subsequent functional expression. Although DNA is generally stable against nuclease degradation, it can still be degraded by nucleases within the cellular environment (Sylvers et al. 2023). In contrast, mRNA does not require nuclear entry for functional expression, as it is transcribed in the cytoplasm into functional protein, thereby bypassing the challenges associated with nuclear delivery. However, mRNAs are highly susceptible to degradation by extracellular ribonucleases (RNases) (Sahin et al. 2014). Similarly, siRNAs function in the cytoplasm and do not need to enter the nucleus, but they are also prone to degradation by RNases, though chemical modifications can enhance their stability (Gavrilov and Saltzman 2012). Like DNA, ASOs also function within the nucleus, requiring nuclear delivery, and are typically chemically modified to improve their stability against nuclease degradation (Angrish and Khare 2023).

This article will explore the physical challenges associated with NA delivery and examine how nanotechnology‐based NA delivery systems provide effective solutions to overcome these obstacles, serving as a viable alternative to traditional nonviral vectors. It will analyze the mechanisms by which these nano‐based systems improve the efficiency and specificity of NA delivery, highlighting recent advancements in the field and their potential implications for therapeutic applications.

2. Concept of NA Therapy

NA therapy involves the use of NAs to edit, replace, or alter the expression of the target gene to manipulate the cellular response to induce therapeutic effects. The main approaches for NA therapy are described as follows (Figure 2).

FIGURE 2.

Strategies in NA therapy. Schematic illustration of four different approaches: (1) Gene editing, a mutated gene is corrected to alter the pathological condition; (2) Gene augmentation, a functional version of the damaged or non‐functional gene is introduced; (3) Gene silencing, suppression of the expression of a target gene; (4) Gene suicide, introduces a cell suicide transgene to cancer cells. Created in BioRender.com .

2.1. Gene Editing

This approach involves the editing of mutated genes in ex vivo, in vitro, or in vivo conditions using programmable nucleases like zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated system (CRISPR/Cas) nucleases (Akbari Kordkheyli et al. 2022). Among these, CRISPR/Cas9 is the latest technology that originated from a bacterial defense mechanism in the early twenty‐first century (Pickar‐Oliver and Gersbach 2019). This technique involves the repair of the DNA double‐strand break (DSB) using programmable nucleases. This technology has significantly transformed gene editing techniques by providing accurate and effective tools for genome editing. Unlike previous techniques like ZFNs and TALENs, the CRISPR/Cas9 simplifies the design of genomic targets and allows for multiplexing, requiring only a target‐specific single‐guide RNA (sgRNA) for each editing task, and eliminating complex protein engineering steps (Akbari Kordkheyli et al. 2022; Li et al. 2023). The CRISPR/Cas9 system comprises two primary constituents: the Cas9 endonuclease enzyme and a guide RNA (gRNA). The gRNA directs Cas9 to the specific DNA sequence, where it causes a double‐strand break, enabling accurate changes to the genome. Gene editing can be carried out using the following strategies. (1) Gene disruption (Carette et al. 2011), primarily used for the treatment of inherited diseases. It involves the direct inhibition of the expression of the target gene. (2) Non‐homologous end joining (NHEJ) gene correction (Su et al. 2016) uses direct knockouts or single base editing. (3) Homology‐directed repair (HDR) based gene editing (Riesenberg et al. 2023) uses homologous recombination to repair DSBs. This approach enables the precise introduction of specific mutations or genetic modifications by providing a synthetic DNA donor as a template for the repair process. The template DNA guides the accurate incorporation of the desired genetic material at the break site. However, the utility of HDR is constrained by its relatively low efficiency, particularly in non‐dividing cells, and the potential for off‐target effects caused by the nuclease activity used to induce DSBs. Additionally, HDR competes with the more error‐prone non‐homologous end joining (NHEJ) pathway, which further limits its effectiveness in many applications (Gray et al. 2022; Song and Stieger 2017).

2.2. Gene Augmentation

Gene augmentation therapy (GAT) involves the introduction of a healthy or functional version of a defective disease‐causing gene or lost gene into the target cell using a delivery vector, which is then expected to reinstate the production of the faulty or missing protein, consequently reversing the disease phenotype (Nóbrega et al. 2020). GAT can be utilized to deliver a variety of NAs of interest including DNA, messenger RNA (mRNA), or oligonucleotides. If the gene intended for delivery is too large for viral vectors, gene editing techniques provide a more effective alternative. Onasemnogene abeparvovec, also known as Zolgensma, is a GAT for Spinal Muscular Atrophy (SMA) (Meyer and Chapman 2022). Approved by the FDA in 2019 for pediatric patients under 2 years old, Zolgensma is designed to address SMA, a genetic disease that leads to progressive muscle weakness. This condition results from an autosomal recessive mutation in the survival motor neuron 1 (SMN1) gene. Zolgensma works by delivering a functional copy of the SMN1 gene to target cells using an adeno‐associated virus (AAV) vector, allowing motor neurons to produce the essential SMN protein. Despite its groundbreaking potential, the cost of Zolgensma is extremely high, with treatment priced around $4.2 million for a one‐time dose. Another notable AAV‐based gene therapy is Luxturna, which was approved by the FDA for the treatment of inherited retinal dystrophy. Both Zolgensma and Luxturna utilize AAV vectors to deliver functional copies of defective genes to target cells, offering innovative treatment options for patients with genetic conditions. While Zolgensma is focused on treating SMA, Luxturna addresses inherited retinal diseases caused by mutations in the RPE65 gene (Meyer and Chapman 2022).

2.3. Gene Silencing

Gene silencing is employed when adding a functional gene to address a disease is ineffective. Rather than introducing a working gene, gene silencing deactivates the mRNA of a specific gene, effectively reducing its functionality to zero. This process typically prevents the expression of the problematic gene, thereby resolving the biological issue in the organism (Huntzinger and Izaurralde 2011). There are two main functional methods to gene silencing: small/short interfering RNA (siRNA) and micro‐RNA (miRNA). siRNA was found to be used as a regulatory point in gene silencing. It has been deemed a post‐transcriptional‐sequence‐specific process (Alshaer et al. 2021). SiRNA therapy has only antagonistic effects and can target un‐druggable targets like many genetic diseases. miRNAs are short RNAs that are key regulators of gene expression; however, the mechanism through which they regulate gene expression is not clear (Carthew and Sontheimer 2009).

2.4. Suicide Gene Therapy

The gene suicide is where a cell suicide‐inducing transgene is introduced to kill the cell. For cancer treatment, this technology can be a breakthrough where viral or bacterial genes can be introduced to cancer cells that can then convert the prodrug to a cytotoxic drug to kill the cell (Duarte et al. 2012; Saeb et al. 2022). However, the tumor targeting ability and time of administration of the prodrug are highly crucial for the successful gene suicide therapy to effectively kill the tumor cells, which can be made possible through targeted delivery via nanoparticle‐based NA delivery systems. The two main strategies used for suicide gene therapy include cytosine deaminase/5‐fluorocytosine and the herpes simplex virus/ganciclovir. Both these techniques have been extensively explored for the treatment of different types of cancers including but not limited to hepatic (Sia et al. 2012), lung (Cramer et al. 2012), colon (Mader et al. 2011), neuroendocrine (Akerstrom et al. 2013), and prostate cancer (Lu et al. 2011).

3. Extra and Intracellular Barriers and the Need for Delivery

To achieve successful NA delivery, there are several biological barriers that need to be circumvented, such as delivery to the target sites, cellular internalization, endosomal escape, and finally accessing the nucleus. These barriers can be categorized into extracellular (EC) and intracellular (IC) barriers (Figure 3). Comprehension of these barriers allows for understanding the importance of delivery systems in NA therapy. The EC challenges that arise for NA delivery include degradation via blood interactions (Liu et al. 2007). The degradation of NAs by nucleases that reside within the blood clears out the NAs from the system, infliction of a response from the innate immune system that causes this clearance (Hamilton et al. 2023). In addition, after overcoming the EC barriers, NAs must overcome the challenges of the IC compartments. NAs are unable to cross the cell's lipid bilayers due to their size, which can range from approximately 20 base pairs to over a million base pairs, as well as their negative charge that causes repulsion against the phosphate heads of the lipid bilayer. Furthermore, NAs, upon entering the IC compartment, face other challenges that could prevent them from entering the nucleus due to their size, entrapment inside endosomes, and susceptibility to degradation by a variety of mechanisms such as endonucleases and lysosomal and proteasomal degradation (Mollé et al. 2022). These biological barriers call for the need for nano delivery systems to maximize the delivery of NAs and thus minimize the effects of these barriers.

FIGURE 3.

This figure depicts a general overview of various barriers for effective NA delivery. Extracellular barriers include enzyme degradation by DNase and RNase, immune cell clearance, nervous system barriers, and other organ and tissue barriers. Intracellular barriers include degradation via intracellular organelles, cellular travel, phagocytosis and exocytosis, and cellular entrance/endosomal escape. Created in BioRender.com .

3.1. Extracellular Barriers

Nucleic acid delivery faces multiple extracellular barriers after systemic administration. They include degradation by nucleases, rapid circulatory clearance, extracellular matrix interactions, and immune recognition. Overcoming these barriers is essential for improving NA stability, bioavailability, and successful cellular uptake.

3.1.1. Systemic Barriers

Systemic travel of NAs poses several challenges including degradation by deoxyribonucleases (DNases) and ribonucleases (RNases) as one of the biggest hurdles causing rapid renal clearance. Intravenous infusion of plasmid DNA in mice has a half‐life of only 10 min (Kawabata et al. 1995). DNase I, an endonuclease, is found primarily in almost all organs and several biological fluids such as blood and urine (Zhang et al. 2024). It degrades circulating DNA and prevents immune response. The DNase II family is primarily found intracellularly in phagosomes or lysosomes and works at lower pH (Lauková et al. 2020; Zhang et al. 2024). RNases are highly effective at degrading extracellular foreign RNAs, including single‐stranded RNA (ssRNA), double‐stranded RNA (dsRNA), and DNA/RNA duplexes (Zhang et al. 2024). RNase A as well as RNase 1–4 are expressed in biological fluids such as blood, cerebrospinal fluid (CSF), and extracellular space, and degrade all types of RNA molecules including mRNA, siRNA, and so on (Bechhofer and Deutscher 2019; Zhang et al. 2024). Nano NA delivery systems designed to protect NAs from degradation face several other challenges. Being considered as foreign materials, nanoparticle‐NA complexes are adsorbed by opsonins and recognized by the mononuclear phagocyte system (MPS) for clearance, leading to reduced transfection efficiencies (Ritz et al. 2015). Additionally, nanoparticles face rapid clearance based on their size. Nanoparticles smaller than 4 nm are quickly excreted through renal filtration, while larger particles are isolated by the spleen and liver, where they are cleared by macrophages (Qin et al. 2023).

Moreover, nanoparticle‐NA complexes often bind to the polyanionic proteoglycans in the extracellular matrix, which can hinder their ability to reach the intended target site, thereby reducing transfection efficiency (Pitkänen et al. 2003). NPs carrying NAs with a diameter of < 5 nm are cleared through the kidneys upon entering the bloodstream, while larger NPs are cleared via the reticuloendothelial system (RES) in the liver (Qin et al. 2023). Nanoparticles are often coated with biocompatible materials like polyethylene glycol (PEG) to extend their circulation time in the bloodstream and minimize detection by MPS (Suk et al. 2016).

3.1.2. Tissue‐Associated Barriers

Even when NAs evade degradation and systemic clearance, they still encounter significant obstacles at the tissue level (Qin et al. 2023). One of these challenges, for example, is overcoming splenic barriers such as the marginal zone and red pulp where particles are phagocytized. The marginal zone is densely populated with macrophages and dendritic cells, which play an important role in recognizing and capturing circulating particles through filtration by sinusoidal endothelial cells and macrophages, continuously searching for foreign substances (Guo and Qian 2024). This splenic clearance remains a significant challenge for nucleic acid therapeutics. In the case of the lungs, NAs must overcome the mucus‐cilia clearance system (MCC) (Qin et al. 2023), a protective mucus layer that traps and removes inhaled pathogens or particles out of the airway, before reaching deep lung tissue (Pangeni et al. 2023). The MCC is a very coordinated mechanism, consisting of goblet cells which secrete mucus and ciliated epithelial cells which propel mucus toward the pharynx, where it is either swallowed or expelled (Pangeni et al. 2023). This overall creates significant challenges for NA therapies as they must navigate through these barriers to reach target cells (Bustamante‐Marin and Ostrowski 2017). Particles smaller than 40 nm have demonstrated improved MCC penetration, whereas larger particles face significant challenges due to steric obstruction with mucin.

Moreover, targeting the central nervous system (CNS) introduces greater complexity due to the blood brain barrier (BBB), a prominent challenge in NA delivery. Since the BBB is semi‐permeable, it restricts about 98% of the molecules from passing through (Padmakumar et al. 2022). In addition to the BBB, the blood cerebrospinal‐fluid barrier (BCSFB) regulates the CSF and its composition. BCSFB is formed by cuboidal epithelial cells, tightly connected by junctions that create a robust barrier, preventing molecules like NAs from entering the cerebrospinal fluid (CSF) of the brain (Ayub et al. 2021). Similarly, the ependyma, which is also regulated by tight junctions, limits the passage of larger molecules from the CSF into the brain (Padmakumar et al. 2022). Nanoparticles based gene and drug delivery systems can overcome various EC barriers by selective targeting to the cells and organs at diseased locations (Chen et al. 2016; Dhull, Wei, et al. 2024; Dhull, Zhang, et al. 2024; Sharma, Sharma, et al. 2020).

Targeting specific tissues is dependent on receptor‐mediated uptake, a selective process which governs internalization of therapeutic agents via specific ligand‐receptor interactions. One major challenge is receptor heterogeneity across the different tissues, as receptor expression varies with cell types, reducing the consistency of NA uptake (Deshmukh et al. 2024).

3.1.3. Immunological Barriers

NAs face significant immunological barriers that can hinder their systemic delivery and intracellular uptake. One challenge is the activation of the innate immune system, where foreign NAs are recognized as potential pathogens. Toll‐like receptors (TLRs), specifically TLR3, TLR7, and TLR8 detect exogenous NAs, leading to the production of pro‐inflammatory cytokines that accelerate clearance and induce systemic inflammation (Subbian and Venketaraman 2024). This recognition reduces therapeutic efficacy and increases the chances of adverse effects. Another barrier is the activation of the complement system, where circulating NAs interact with complement proteins, leading to their clearance by phagocytic cells in the liver and spleen (Booker et al. 2025). This limits the circulation of NA therapies and reduces the ability to reach target tissues. Opsonization occurs when plasma proteins such as immunoglobulins and complement factors coat NA complexes, making them susceptible to macrophages for uptake, reducing bioavailability (van den Berg et al. 2021).

3.2. Intracellular Barriers

After NAs successfully overcome the extracellular barriers such as tissue obstacles and systemic clearance, they face new challenges ultimately having to overcome IC barriers. The first IC challenge is accessing the cell. NAs, including several types of DNA, RNA, or oligonucleotides, generally have a negative charge due to the phosphate backbone. Similarly, the lipid bilayer of the cell membrane contains a negative charge, making diffusion across the lipid bilayer improbable (Liu et al. 2007). Additionally, the size of the NAs further complicates entry into the cell, as plasmid DNA or certain RNA can be too large to pass through channel proteins (Liu et al. 2007). However, NA‐conjugated NPs can enter the cell primarily through the process of endocytosis, which is further broken down into phagocytosis and pinocytosis (Hu et al. 2024). Phagocytosis is a specialized method of endocytosis that engulfs and clears particles larger than 0.5 μm and only occurs in phagocytic cells such as macrophages and neutrophils (Hu et al. 2024). Upon engulfment, the particles form phagosomes that fuse with lysosomes to degrade the material that is engulfed. However, pinocytosis is more common and occurs in almost all cells and can be subdivided into four types, including clathrin‐dependent endocytosis, macropinocytosis, clathrin‐and‐caveolae‐independent endocytosis, and caveolae‐dependent endocytosis. Studies reveal that the preferred method of endocytosis varies for different nanoparticles. For example, caveolae‐mediated endocytosis is the preferred method for lipid nanoparticles (LNPs) (Qin et al. 2023).

Regardless of the specific endocytosis mechanism, a significant challenge in NA delivery is endosomal escape. Once inside the cell, vesicles fuse with early endosomes and progress through the endocytic pathway, maturing into late endosomes. If these vesicles fail to escape the endosomes, they eventually fuse with lysosomes, which possess an acidic pH below 5. This harsh environment typically leads to the degradation of the vesicle contents, including the NAs, hindering their therapeutic efficacy (Hu et al. 2024; Qin et al. 2023). Additionally, late endosomes can also participate in autophagy, where they fuse with autophagosomes to form amphisomes, which subsequently follow the lysosomal pathway for degradation of NA cargo. However, if this cargo is not degraded by lysosomes or through autophagic processes, it can be exocytosed. In this case, the endosome fuses with the plasma membrane, releasing the cargo back into the extracellular space (Vermeulen et al. 2018). This can occur at multiple stages of endosome maturation. Early endosome may be recycled to plasma membrane or undergo maturation to a multivesicular body then fuse with the plasma membrane, late endosome can be recycled via the endoplasmic reticulum and Golgi route then fuse with plasma membrane, or lysosome can release non‐degraded contents into extracellular space (Hu et al. 2024). These consequences further increase the challenges of endocytosis. Nano delivery systems can overcome these challenges of endosomal escape. Nanoparticles have been shown to disrupt the negatively charged membranes of endosomes by interactions with positively charged nanoparticles. Polyamine cationic nanoparticles have been shown to undergo endosomal escape via “proton sponge effect” (discussed in Section 3) (Hoekstra et al. 2007; Rayamajhi et al. 2020).

Additionally, if endosomal escape is achieved, navigation of NAs within the cytoplasm places them at risk for degradation by enzymes such as nucleases, proteases, or lipases. These barriers are similar to the ones in EC barriers as the families are similar in reference to DNase and RNase families (Zhang et al. 2024). Moreover, navigating the cytoplasm poses challenges for the effective transport of NAs due to the complex architecture of the cell. This complexity includes the cytoskeleton, composed of microtubules and actin filaments, which not only provide structural support but also affect intracellular transport, often causing delays or misdirection. Additionally, several cellular organelles such as the endoplasmic reticulum and Golgi apparatus further complicate the path for the NAs (Juliano and Carver 2015). Therefore, traveling through the cytosol is ultimately highly dependent on the size of the NAs, where smaller NAs such as miRNA or siRNA may navigate the cytosol easier than larger NAs such as mRNA or pDNA (Hu et al. 2024).

Different types of NA therapeutics, such as DNA, mRNA, siRNA, and antisense oligonucleotides (ASOs), present unique delivery challenges within the cells. These challenges include, but are not limited to, efficient nuclear entry and susceptibility to nuclease degradation. Furthermore, delivering NAs to the nucleus presents a significant challenge due to the highly selective nature of the nuclear envelope. Composed of an outer and inner membrane, separated by perinuclear space, the nuclear envelope restricts passage to molecules based on specific size, charge, or signaling properties through its nuclear pore complexes. This high level of selectivity poses a significant barrier to the efficient transport of NAs. For example, molecules less than 9 nm can diffuse through the nuclear pore complex while larger ones need active transport mechanisms to pass (Durymanov and Reineke 2018). This poses a challenge since therapeutic DNA can be up to kilobase pairs, which is roughly 340 nm (Durymanov and Reineke 2018).

4. Nano NA Delivery Systems

In vivo NA delivery faces several obstacles that must be overcome to fully realize the therapeutic potential of NA‐based therapeutics. The selection of an appropriate delivery vehicle is crucial to ensuring successful NA delivery. An ideal vector should overcome both external and internal barriers, such as shielding NAs from enzymatic degradation, promoting efficient intracellular uptake and endosomal escape, and exhibiting low toxicity to minimize off‐target effects. Achieving a balance between these functions is key to optimizing therapeutic outcomes and advancing the application of nucleic acid therapeutics in clinical settings. Nanoparticles can be designed to increase the protection of genetic material against degradation, achieve targeted delivery, increase stability and blood stream circulation, and reduce toxicity (Chen et al. 2016). Ultimately, the diverse toolbox offered by nanoparticles with different properties contributes to their success and importance in NA therapy. Each type of nanoparticle brings unique properties and advantages, creating versatile platforms tailored for enhanced NA delivery, allowing for more precise, efficient, and targeted delivery of NAs to affected cells. In this section, we provide insight into several nanoparticles, including bioengineered nanoparticles, LNPs, and polymeric nanoparticles, utilized for NA delivery purposes.

4.1. Bioengineered Nanoparticles

Bioengineered nanoparticles for NA delivery are a promising tool in modern medicine, offering a highly efficient and targeted approach to treat genetic disorders. Their tunable size, surface chemistry, and biocompatibility make them ideal for enhancing cellular uptake, protecting genetic material from degradation, and enabling precise control over gene expression. These nanoparticles are designed to mimic the sophisticated strategies nature employs for packaging and delivering genetic materials. By drawing inspiration from the inherent structural and functional principles of biological systems, such as viruses and cells, bioengineered nanoparticles are designed to enhance the precision, targetability, and efficiency of NA delivery. This approach aims to improve the safety and effectiveness of gene therapies by reducing off‐target effects and minimizing immune responses, ultimately providing a more reliable and targeted treatment method for a wide range of diseases.

4.1.1. Virus Like Particles

Virus‐like particles (VLP) are self‐assembling symmetrical particles from viral structural proteins that lack viral genetic material (Lamarre and Ryadnov 2011) (Figure 4). VLPs have several advantages that have allowed widespread clinical use in vaccinology, including their good safety profile, stability, assembly flexibility, and symmetry (Kwon and Giessen 2022; Lamarre and Ryadnov 2011; Ruzzi et al. 2023; Zeltins 2013). In the past several decades, VLPs have extensively been used for applications in vaccinology, such as Hep B and HPV, as structural proteins and surface antigen epitopes can induce strong cellular and humoral immune responses (Ruzzi et al. 2023). Many different types of viruses and glycoproteins can be used as VLPs, which allows tuning of cellular tropism to avoid unwanted targeting of tissues. Additionally, given their small size, VLPs can cross the BBB easily and are very biocompatible. As VLPs can transduce mammalian cells and release their cargo, they offer a promising avenue for NA delivery. To date, human clinical applications have been mostly limited to vaccinology, but several preliminary studies have explored their ability to deliver Cas9 nuclease to edit genomes in murine studies. Engineered VLP containing Cas9 ribonucleoprotein has been explored to obtain therapeutic levels of gene editing in various tissues, including the liver, retina, and brain (Banskota et al. 2022). Another VLP has been engineered from murine leukemia virus‐like particles containing Cas9‐sgRNA ribonucleoprotein to edit transcripts with in vivo models of mice liver and mouse embryos (Mangeot et al. 2019).

FIGURE 4.

Schematic representation of bioengineered nanoparticles for NA delivery applications. Figure demonstrates the representation of virus‐like particles, multifunctional protein nanocages, and ferritin nanocages. Created in BioRender.com .

Specific targeting of tissues can be obtained by changing glycoproteins, but much of the work remains limited to the pre‐clinical phase. Efficient delivery of VLPs remains a concern as the delivery vehicle needs to be stable to prevent degradation prior to reaching the intended cellular target, but directly targeted injections (i.e., intraocular or intrahepatic) may provide an avenue for high efficacy (Raguram et al. 2022). While overall promising, much of the work remains limited to the pre‐clinical phase and will require extensive validation before translation into clinical trials.

4.1.2. Ferritin Cages

Ferritin‐based nanocarriers are derived from subunits of heavy chain ferritin (HFn) and light chain ferritin (LFn) that carry iron (Figure 4). Prior groups have modified ferritin subunits to carry alternative cargo molecules for a wide variety of applications, including tumor therapeutics, imaging compounds, and genetic material (Song et al. 2021). Specifically, the HFn nanocages have transferrin receptor 1 (TfR1) on their exterior surface, allowing tuned targeting of tumor and endothelial cells (Song et al. 2021). This has allowed several groups to develop therapeutic ferritin‐based nanoparticles, including chemotherapy agents such as cisplatin and doxorubicin (Liang et al. 2014; Pontillo et al. 2016).

Similar to VLPs, the clinical application of ferritin cages has been limited to vaccine development. Ferritin nanocages can be tuned to display specific antigens and have previously been demonstrated to induce a potent immune response (Lee et al. 2022; Rodrigues et al. 2021). Early clinical trials have been started for ferritin‐based nanoparticle approaches for influenza, Epstein Barr Virus, and SARS‐CoV‐2. However, given their excellent biocompatibility and promising drug delivery platform, ferritin‐based nanoparticles have also been explored for siRNA delivery. Yuan et al. created a cationic HFn variant to deliver siRNA to glioma cells past the BBB (Yuan et al. 2022). Another group loaded bisdemethoxycurcumin into ferritin nanoparticles to treat Alzheimer's‐related inflammation (Gagliardi et al. 2022).

While promising, further work will be needed before the clinical translation of ferritin nanoparticles for NA delivery. Several challenges remain in the manufacturing process for ferritin nanoparticles, including particle heterogeneity, cargo loading efficiency, and antigen interference from inter‐subunit interactions (Rodrigues et al. 2021).

4.1.3. Lumazine Synthase

Lumazine synthase is an enzyme responsible for the biosynthesis of riboflavin. The protein cage of lumazine synthase, isolated from the bacterium Aquifex aeolicus, has been utilized as a scaffold for drug delivery and NA therapy, given its biocompatibility, facile reproduction, and high tolerance to modifications (Azuma et al. 2018; Edwardson et al. 2018; Ra et al. 2014). Edwardson et al. were able to load siRNA into the lumazine synthase nanocage and deliver it to mammalian cells for reduction in gene expression (Edwardson et al. 2018). Similarly, another study reported tuning peptides into the nanocage structure to target endothelial cells and hepatocellular carcinoma (Min et al. 2014). However, lumazine synthase was also found to recruit serum antibodies when displaying antibody binding sites, suggesting that the utility of lumazine synthase nanocages may be limited to in vitro applications (Levasseur et al. 2021).

4.1.4. Protein Nanocages

Protein nanocages refer to self‐assembling sub‐proteins that form a cage and have been utilized as potential carriers for NA delivery (Figure 4). Protein nanocages can be derived from naturally occurring biological systems such as VLPs, lumazine synthase, and ferritin. Alternatively, protein nanocage subunits can be inorganically synthesized to create hybrid nanocages. These systems have three different components, specifically the interior, inter‐unit, or exterior regions, that can be tuned to carry a specific genetic load and increase target specificity (Choi et al. 2018; Kwon and Giessen 2022).

4.1.5. Extracellular Vesicles and Exosomes

Exosomes are small extracellular vesicles excreted by cells ranging from 50 to 500 nm (Cecchin et al. 2023; Chen et al. 2021). Exosomes are critical in modulating physiological pathways as transporters via several different mechanisms, including but not limited to endocytosis, phagocytosis, and membrane fusion (Cecchin et al. 2023; Chen et al. 2021) (Figure 5). Surface markers on exosomes are dependent on the originating cell, demonstrating a wide variety of targeting markers and tissue tropism (Wallen et al. 2023). Target surface markers can further be modified synthetically by functionalizing peptides for better affinity (Liang et al. 2020).

FIGURE 5.

Schematic representation of the loading of exosomes with nucleic acid materials, mechanisms of their intracellular uptake, target surface markers including peptides, aptamers, and antibodies, and synthetic challenges leading to limited clinical clearance. Created in BioRender.com .

Exosomes have previously been loaded with a variety of different gene therapy agents, including siRNA, miRNA, and proteins such as CRISPR/Cas9 (Lu et al. 2023; Wallen et al. 2023). Kobayashi et al. purified exosomes from omental fibroblasts of ovarian cancer patients and loaded them with tumor suppressor (TS) miRNAs (Kobayashi et al. 2020). Treatment with the TS miRNAs loaded exosomes on ovarian cell lines demonstrated increased expression of TS miRNA within cells and suppressed c‐MET expression, which inhibited cell proliferation. Similarly, Zhao et al. developed a new exosome containing siRNA to inhibit the growth of metastatic breast cancer cells (Zhao et al. 2020).

Several groups have also loaded exosomes with CRISPR/Cas9 plasmids and RNA to enable gene editing. Kim et al. loaded CRISPR/Cas9 plasmids to suppress poly (ADP‐ribose) polymerase‐1 (PARP‐1) in ovarian cancer to induce apoptosis and increase sensitivity to cisplatin (Kim et al. 2017). Usman et al. utilized exosomes from red blood cells loaded with HA‐tagged cas9 mRNA to gene edit leukemia cells (Usman et al. 2018).

While exosomes hold significant promise in pre‐clinical studies, there are several challenges. Namely, issues in production with heterogeneous loading efficiency, lack of standardization, and rapid clearance from circulation need to be sorted prior to widespread clinical translation (H. Chen et al. 2021). As the cellular origin of exosomes is important for tropism, precise scalable purification methods will need to be developed prior to translation.

4.2. Peptide Nanoparticles

Peptides are composed of amino acids typically ranging from 500 to 5000 kDa and serve an essential role in many physiological processes involving cellular signaling (Wang et al. 2022). Following characterization of the insulin peptide in the 1920s, over 90 different FDA‐approved peptide medications are now available in the market (Fosgerau and Hoffmann 2015; Wang et al. 2022). Given their excellent biocompatibility, existing facile manufacturing methods, and relatively low toxicity, significant interest has turned to utilizing peptides as gene delivery vehicles. Peptide molecules are also relatively small compared to other synthetic compounds, uniquely positioning them for intracellular delivery.

For successful peptide mediated NA delivery, the peptides need to form a nanoparticle that allows protection of genetic material from degradation, immunogenicity, and opsonization. To do so, the inherent properties of each amino acid can be utilized to design the nanoparticle of choice. For binding and condensing DNA, arginine and lysine are classically used given they are cationic amino acids and can condense DNA via electrostatic forces. A study by Mann et al. observed that the ideal homopeptide length for both arginine and lysine was around 16 units for DNA condensation and release (Mann et al. 2011). To increase stability, hydrophobic amino acids such as leucine and valine can be incorporated to create amphiphilic cationic derivatives. This ultimately allows for more effective NA delivery by allowing these compounds to go through the cell membrane (Liu et al. 2010). In one example, Yu et al. incorporated hydrophobic N‐acetyl‐L‐valine pendants in guanidine rich helical peptides to increase gene transfection by up to 6 time in murine models (Yu et al. 2021). Histidine has also been widely utilized given its unique pH responsive properties. Its amphiprotic properties allow protonation within the endosome, which can lead to rupture of the endosome and release of material into the cytoplasm via the “proton sponge effect” (Hooshmand et al. 2022). Lee et al. modified polyamidoamine dendrimers with histidine and arginine to increase NA delivery of heme oxygenase‐1 in brain reperfusion animal models (Y. Lee et al. 2021).

Additionally, to improve penetration through the cellular plasma membrane, specific protein transduction domains (PTDs) or cell penetrating peptides (CPP) have been identified. One of the first CPPs (YGRKKRRQRRR) was identified from the trans‐activator of transcription (TAT) Human Immunodeficiency Virus (HIV) in 1989 (Green et al. 1989). In one example, Schwarze et al. fused the TAT protein to a beta‐galactosidase protein and successfully delivered the protein within mouse models via intraperitoneal injection (Schwarze et al. 1999). Since then, a significant number of tissue and cell specific CPPs have been identified and utilized by various groups for targeted NA therapy (Taylor and Zahid 2020). Tuttolomondo et al. used a human protein derived DMBT1 CPP to deliver siRNA to tumor cells in vitro (Tuttolomondo et al. 2017). Similarly, Arap et al. utilized phage display to identify CPP specific to tumor blood vessels. Following this, the group conjugated the CPP to doxorubicin, a chemotherapy agent, to target tumor in a xenograft breast cancer mouse model (Arap et al. 1998). For further fine tuning of cellular tropism, several groups have built specific peptide fragments conjugated systems to allow for targeted NA therapy. Wang et al. constructed an arginine rich motif conjugated to a hepatocellular carcinoma specific peptide fragment to deliver siRNA in mice models (Wang et al. 2014). Similarly, Ren et al. developed a peptide GE11‐polyethylene glycol‐polyethylenimine (GE11‐PEG‐PEI) to target epidermal growth factor receptor (EGFR) expressing laryngeal cancer (Ren et al. 2015).

Ultimately, peptide‐mediated NA therapy holds significant promise, but there are several remaining challenges. Namely, peptides are less stable due to degradation by enzymes and lack specificity compared to antibodies, but they have significant advantages with existing production methods, low toxicity, and a tunable approach allowing for tissue tropism (Kang et al. 2019).

4.3. Lipid Nanoparticles (LNPs)

Lipids are amphipathic molecules containing a hydrophilic head, hydrophobic tail, and an intermediate that connects the two (Figure 6). The idea of LNPs was developed in the late 1960s when it was discovered that lipid bilayer vesicles spontaneously form in water, which led to liposomes being used in the delivery of mRNA (Hou et al. 2021; Mashima and Takada 2022; Tenchov et al. 2021). The use of LNPs has extended past NA delivery, where they are also used in medical imaging, nutrition, agriculture, and are an important component of COVID‐19 mRNA vaccines (Tenchov et al. 2021). LNPs typically range from 50 to 100 nm in diameter, exhibit a spherical shape, and possess a micellar structure. They are characterized by enhanced kinetic stability and a more rigid morphology compared to other nanoparticle systems. LNPs enable highly efficient encapsulation, offering robustness and scalability in their manufacturing process. Additionally, they exhibit excellent stability, with a shelf life of approximately 1 year when stored at 4°C, making them a well‐established and mature technology for therapeutic applications (Cullis and Hope 2017). LNPs are typically composed of ionizable lipids, cationic lipids, PEG‐conjugated lipids, phospholipids, and cholesterol. Among these, ionizable lipids play an important role in facilitating intracellular delivery through the stabilization of NA complex and promoting endosomal escape in acidic environments (Mashima and Takada 2022; Mendes et al. 2022). Recent advancements are focused on the optimization of ionizable lipids for better biocompatibility and reduced systemic toxicity. Furthermore, branched fatty acid‐based novel ionizable lipids have shown improved stability in lipid self‐assembly, nucleic acid binding, and phase behavior, which may help in their overall stability. Pawlowska et al. investigated these novel ionizable lipids, showing that lipid head‐group size and alkyl chain flexibility can significantly impact DNA binding capacity (Pawlowska et al. 2024). These findings show how modifying the structure of ionizable lipids can be used to optimize NA delivery systems with improved therapeutic outcomes.

FIGURE 6.

Illustration of lipid Nanoparticles (LNP) containing a composition of cationic ionizable lipids, PEG‐Lipids, phospholipids, and cholesterol used primarily for delivery of RNA. PEG‐Lipids make up a small portion (1%–3%) of the overall LNP; however, the addition of PEG‐Lipids allows for greater LNP stability, half‐life in the bloodstream, and a decrease in NP aggregation. Created in BioRender.com .

Another example of modifications in the formulation of LNPs is the application of C‐24 alkyl phytosterols to improve the intracellular delivery efficiency. Kimura and Harashima found that C‐24 alkyl phytosterols‐containing LNPs exhibited improved diffusivity and stability when compared to cholesterol‐based LNPs. They observed that these modifications influenced their biodistribution and increased organ targeting when used in applications for NA delivery. This aligns with increasing interest in optimizing lipid composition for optimal therapeutic effect by regulation of NP interaction with biological membranes.

Tissue‐targeting is an emerging field of interest in LNP research. Ligand‐functionalized LNPs have been investigated to increase organ‐specific delivery. In the liver, GalNAc (N‐acetylgalactosamine) functionalized LNPs have been widely studied for targeted siRNA‐based therapeutics, leveraging hepatocyte‐specific asialoglycoprotein receptors (ASGPRs) for increased cellular uptake and gene silencing activity (Debacker et al. 2020). These advances make GalNAc functionalized LNPs promising for the treatment of liver diseases. In addition, efforts have been made to enhance LNP penetration across the BBB for neurotherapy. Peptide‐conjugated LNPs have been employed as BBB shuttle peptides or cell‐penetrating peptides to facilitate endothelial barrier‐mediated transcytosis to improve efficiency for neurodegenerative and brain tumor therapies (Kristensen et al. 2020; Wu and Angelova 2023). These approaches reflect the growing need to optimize LNP formulations for maximum tissue selectivity and therapeutic effects.

4.3.1. Cationic and Ionizable Lipids

Cationic lipids contain heads with permanent positive charges, which lead to higher efficiency in NA encapsulation compared to the systems that are neutral (Hou et al. 2021; Mashima and Takada 2022). 1,2‐Di‐O‐octadecenyl‐3‐trimethylammonium propane (DOTMA) is a quaternary ammonium lipid used in mRNA delivery and is commercialized as Lipofectin combined with DOPE. Spleen‐targeting DOTMA has been developed as a cancer vaccine (Hou et al. 2021). Although cationic lipids offer higher efficiency due to their charged head groups and have shown promise in therapeutic applications, their use is limited by reduced circulation times and increased toxicity. The positive charge can disrupt cellular interactions and negatively interact with biological molecules, leading to potential toxicity concerns (S. Wu et al. 2024). More recently, ionizable lipids have emerged as crucial components in LNP formulations. One of the first ionizable lipids, 1,2‐dioleoyl‐3‐dimethylammonium propane (DODAP), demonstrated the ability to achieve high encapsulation efficiencies when combined with other lipids in the presence of ethanol (Semple et al. 2001). Ionizable lipids consist of three parts: the head, intermediate, and tail; and the ionizable head groups can be composed of amine, guanidine, and heterocyclic groups (S. Wu et al. 2024). Ionizable lipids are pH sensitive, with a pKa of 6–7; therefore, being protonated at lower pH and are neutral at physiological pH, providing a great advantage for NA delivery due to decreased interactions with anionic blood cell membranes, improving biocompatibility and increasing blood circulation. Advantageously, the positive charge at low pH can interact with negative charges in the endosomal membrane, destabilizing the endosome and ultimately promoting endosomal escape and the release of NAs (Figure 6). Moreover, incorporating biodegradable lipids, such as carboxylic esters that can be cleaved by esterases, enhances the tolerance of LNPs by promoting faster metabolism, quicker clearance, and reduced side effects (Hou et al. 2021; Wu et al. 2024). Zwitterionic ionizable lipids have also been employed in NA delivery, particularly for mRNA delivery (Hou et al. 2021). LNPs that are composed of these zwitterionic lipids and three hydrophobic tails can form a cone structure within the acidic environment of the endosome, facilitating hexagonal phase transformations. This transformation enables efficient endosomal escape, leading to faster and more effective protein expression or genome editing (Hou et al. 2021; Liu et al. 2021).

4.3.2. Other Lipids

PEG‐lipids were originally utilized in liposomal systems in order to extend the half‐life in circulation. This occurs due to the steric barrier produced by PEG that prevents it from binding to opsonins, which would lead to clearance by RES (Samaridou et al. 2020). PEG‐lipids are important components of LNPs, located on their surfaces and conjugated with hydrophobic tails via connecting intermediates, with the lipid domain facing down into the particle, and the PEG domain extending out of the surface (Samaridou et al. 2020; Wu et al. 2024). Although the PEG‐lipids constitute only around ~1.5 mol% of the composition of LNPs, these provide several benefits and are useful in preventing the binding of proteins, decreasing NP aggregation, improving LNP stability, increasing half‐life in the bloodstream, and determining LNP size in manufacturing (Hou et al. 2021; Samaridou et al. 2020; Wu et al. 2024). PEG‐lipids in LNPs are commonly used for RNA delivery; for example, PEG2000‐C‐DMG is used for the formulation of the first siRNA drug Onpattro approved by the FDA (Hou et al. 2021; Wu et al. 2024). Various other helper lipids, such as phospholipids and cholesterol, are used in LNP formulations to provide storage and plasma stability. Phospholipids, generally, are composed of a phosphate group with two hydrophobic tails linked together via glycerol. Phospholipids assist in endosomal escape and aid in enhancing the efficiency of mRNA delivery (Tenchov et al. 2021). For example, 1,2‐distearoyl‐sn‐glycero‐3‐phosphocholine (DSPC) is a phosphatidylcholine characterized by its saturated fatty acid chains, which confer a cylindrical shape to its molecular structure. This geometry, coupled with its exceptionally low melting temperature of around −54°C, enables DSPC to form a stable lamellar phase. This property is crucial for the stabilization of LNPs. DSPC was used in mRNA‐1273 and BNT162b2 COVID‐19 vaccines (Hou et al. 2021). 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoethanolamine (DOPE), a comparatively unsaturated lipid, is a phosphoethanolamine containing two unsaturated tails, a conical shape, and a melting temperature of about −30°C. DOPE takes on an inverted hexagonal shape, which destabilizes the membrane of endosomes and assists in the endosomal escape of LNPs (Hou et al. 2021). Furthermore, cholesterol is a class of terpenoid lipids and is an essential precursor for the synthesis of several steroids (Wu et al. 2024). Cholesterol is considered an essential component of LNPs, used at a concentration of about 30%–40% of total lipids and assists in maintaining the LNP in a rigid form, enhancing its stability and increasing the efficiency of fusion with the cell membrane (Samaridou et al. 2020; Wu et al. 2024). For example, cholesterol analogues with C‐24 alkyl phytosterols increase the in vivo delivery efficacy of LNPs in mRNA formulations (Patel et al. 2020). For LNPs, the length of hydrophobic tails of cholesterol, the flexibility of sterol rings, and the polarity of hydroxyls are the determinants of delivery efficacy (Hou et al. 2021). Research shows that cholesterol analogs produced via oxidation and esterification can be delivered to diverse cells such as endothelial cells, hepatocytes, and macrophages. However, LNPs with cholesteryl oleate show a greater selectivity for liver endothelial cells than hepatocytes (Hou et al. 2021; S. Wu et al. 2024).

4.4. Polymeric Nanoparticles (PNPs)

Recent advances in polymer science and nanotechnology have led to the development of a diverse array of polymeric nanoparticles (PNPs) that can effectively overcome physiological barriers for successful NA delivery (van den Berg et al. 2021). Although PNPs are less commonly used in clinical settings compared to LNPs for NA delivery applications, they offer unique properties that make them highly advantageous for targeted delivery to specific organs, tissues, and cells. There is a plethora of polymers that show promise as candidates for NA delivery, including poly(amidoamine) dendrimers, poly(L‐lysine), poly(ethyleneimine) and natural polymers such as chitosan and dextran, to name a few (Rai et al. 2019) (Figure 7). PNPs have been researched extensively due to their properties, which range from biodegradability, efficient synthesis, low toxicity, and decreased cost of production (Rai et al. 2019; Sung and Kim 2020). Their versatility and ability to be engineered for NA delivery applications present exciting opportunities for advancing NA therapy strategies.

FIGURE 7.

Representation of polymeric nanoparticles‐based non‐viral gene delivery vectors. These include poly(amidoamine) dendrimers, poly (L‐lysine), poly(ethyleneimine), chitosan and dextran. Created in BioRender.com .

4.4.1. Dendrimers

Dendrimers are tree‐like, hyperbranched, and monodispersed macromolecules being widely used for drug and NA delivery applications, with promising clinical outcomes (Dhull et al. 2023; Dhull, Wei, et al. 2024; Dhull, Zhang, et al. 2024; Rani et al. 2024; Sharma et al. 2016). Dendrimers contain repeating branched spherical structures composed of an inner core, branching units, and a high density of surface groups. Dendrimers offer several advantages over other PNPs, including nanoscale size, control over structure, shape, physicochemical properties, and in turn, their physiochemical properties. The surface cationic groups of dendrimers allow encapsulation with NAs via electrostatic interactions, ensuring their protection from degradation. However, the NA delivery efficiency of dendrimers is governed by several attributes, including dendrimer structure, surface groups, size, generation, and other physicochemical features (Chis et al. 2021). Among various dendrimers, polyamidoamine (PAMAM) dendrimers are the most widely explored for NA delivery applications (Tarach and Janaszewska 2021). Surface‐engineered dendrimers with various ligands, including lipids, peptides, sugars, amino acids, etc., have been reviewed earlier for NA delivery applications (Yang et al. 2015).

4.4.2. Poly(L‐Lysine)

Poly(L‐lysine) (PLL) is a biodegradable, cationic polypeptide composed of lysine monomers and is one of the first PNPs being used for NA delivery (Sung and Kim 2019; Thomas et al. 2019; Wu and Wu 1987). Since PLL is a cationic peptide, it is capable of being protonated, thus allowing for establishing complexes with DNA for NA delivery (Manouchehri et al. 2021). PLL can be synthesized in several conformations and molecular weights, including linear, hyper‐branched, and dendritic, where each has different safety profiles. Dendritic PLL has shown greater gene transfection as opposed to linear PLL (Rai et al. 2019; Thomas et al. 2019). However, PLL/NA complexes exhibit low transfection efficiency due to slow endosomal escape (Zhang et al. 2010). PLL modifications have shown promise in NA delivery; for example, Kodama et al. synthesized dendrigraft PLL (DGL) conjugated with γ‐polyglutamic acid (γ‐PGA) which resulted in high DNA transfection in organs such as the liver and lungs (Kodama et al. 2014).

4.4.3. Poly(Ethylenimine)

Polyethylenimine (PEI) was introduced in 1995 as a synthetic, cationic polymer designed for NA delivery. It is a widely explored PNP for NA delivery applications due to its exceptional ability to condensate and stabilize NAs. The presence of protonable amino groups at every third position, PEIs exhibit a high cationic charge density, which results in the condensation of nucleic acids into complexes. These polyplexes demonstrate remarkable stability at room temperature and when stored long‐term as frozen complexes (Höbel et al. 2008). Interestingly, PEIs have been demonstrated to be some of the most effective non‐viral agents for gene transfer in vitro (Boussif et al. 1996). PEI and nucleic acids can be conveniently and reproducibly complexed together simply by mixing. The resulting complexes exhibit a “spaghetti‐meatball‐like” structure, where the spaghetti symbolizes the elongated polymer and the meatballs represent the encapsulated nucleic acid cargo (Kneuer et al. 2000). The cellular uptake of PEI polyplexes occurs through the interaction of their positive charge with negatively charged cell membranes leading to endocytosis. This is followed by the endosomal escape by following mechanisms: proton sponge effect and umbrella effect. These mechanisms cause the swelling of endo/lysosomal compartments that eventually create nanoholes in the endosomal membrane (Vaidyanathan et al. 2016). High molecular weight (HMW) linear PEIs have been developed into commercial transfection reagents such as jetPEI. However, the high gene transfection efficiency of HMW PEIs is typically accompanied by significant cytotoxicity (Chollet et al. 2002). This cytotoxicity, particularly at higher doses, poses a major obstacle to the broader use of HMW PEIs in NA therapy.

4.4.4. Chitosan

Chitosan is an FDA approved, natural cationic carbohydrate polymer that is derived from chitin and has gotten a lot of attention as a PNP for NA delivery (Dong et al. 2024; Sung and Kim 2020; Thomas et al. 2019). Chitosan is a linear copolymer which is composed of copolymers of D‐glucosamine and N‐acetyl glucosamine units linked via β‐(1,4) glycosidic linkages. Due to chitosan being natural, it has many favorable characteristics that catch the attention of scientists, such as biocompatibility, biodegradability, nontoxicity, and the aqueous derivatives of chitosan; chitosan salts are particularly attractive for their water‐soluble properties (Sung and Kim 2020). The amino groups on chitosan get protonated at lower pH, enabling complexation with negatively charged NAs. Initial studies with chitosan vectors were more focused on high molecular weight chitosan (> 300 kDa) due to its greater stability and enhanced endo/lysosomal protection. However, high molecular weight enables stronger mutual interactions that result in slower NA release after endosomal escape (Sato et al. 2001). Recently, low molecular weight chitosan has garnered significant attention due to low viscosity, better solubility at physiological conditions, and convenience for chemical modifications. Moreover, comparatively weak interactions with NAs enable ease of regulation between the stability of complexes and transfection efficiency. Similar to other cationic polymers, chitosan also promotes endosomal escape via the proton sponge method. Despite showing potential for NA delivery in preclinical studies, there is a lack of clinical data on the use of chitosan for NA delivery applications. This is primarily due to concerns about toxicity and immunogenicity caused by heavy metal impurities present in chitosan (Bezrodnykh et al. 2020).

4.4.5. Dextran

While cationic polymers are promising candidates for NA delivery, their clinical application is hindered by challenges such as serum instability and cytotoxicity. In contrast, dextran, a non‐cationic, biodegradable carbohydrate polymer, offers a superior alternative due to its excellent physicochemical properties. Its biocompatibility, biodegradability, and non‐immunogenic nature make it an attractive option for various medical applications, including NA delivery. Dextran's structure, rich in hydroxyl groups, allows for easy chemical modification to introduce cationic charge, thereby enhancing transfection efficiency and promoting effective endosomal escape. Dextran is modified with cationic residues such as peptides (Tang et al. 2014), PEI (Jiang and Salem 2012), agmatine (Jianhai Yang et al. 2012), spermine (Eliyahu et al. 2005) and so on, to promote electrostatic interactions with negatively charged NAs. Dextran conjugated PEI demonstrated excellent transfection efficiency while reducing the toxicity associated with PEI and enhancing its serum stability (Jiang and Salem 2012). However, the physiological fate of dextran remains incompletely understood, necessitating further optimization of dextran‐based nanosystems for their effective use in NA therapy.

4.4.6. Other Biodegradable Polymers

Gelatin is a natural biodegradable polymer that has been widely used for gene delivery due to its high biocompatibility (Madkhali et al. 2019). Youngren and colleagues designed STAT6 siRNA loaded gelatin nanocarriers for inhibition of the STAT6 gene, a cell‐proliferation related gene, for the treatment of cancer. The formulation successfully silenced STAT6 gene expression in A549 cancer cells, subsequently killing them (Youngren et al. 2013). Albumin is also used in NA delivery in combination with cationic polymers due to the lack of inherent positively charged functionalities. For instance, Syga and coworkers investigated the effect of incorporating bovine serum albumin (BSA) into linear polyethylenimine (LPEI) polyplexes for plasmid DNA delivery, focusing on how albumin influences transfection efficiency in HeLa cells (Syga et al. 2016). The order of albumin incorporation, either before or after polyplex formation, was found to significantly affect the uptake and transfection efficiency.

5. Target Diseases and Routes of Administration

Diseases with mutations in a single gene are particularly well suited for NA therapy. In this section, we discuss not only the routes of administration for nano‐based approaches for NA delivery (Figure 8) but also place an emphasis on nano delivery technologies in early human clinical trials (Table 1). While almost all current FDA‐approved medications for gene therapies are viral vector‐based due to their transduction efficacy, they are limited by concerns with immunogenicity, toxicity, and cost. Currently, most early phase trials for non‐viral vector‐based therapies are limited to oncological applications. While not exhaustive, we divide target diseases into three different categories and discuss their associated routes of administration: oncologic, metabolic, and neurologic.

FIGURE 8.

Schematic diagram representing various routes of administration for nano NA delivery systems. Created in BioRender.com .

TABLE 1.

Select clinical trials of NA delivery with nanoparticles.

Nanoparticle	Gene agent	Disease	Gene Target	Route of Administration	Phase	CT.gov
Oncologic
Lipid Nanoparticle (Oncoprex)	Quaratusugene ozeplasmid (Reqorsa)	Lung Cancer (small cell and non‐small cell)	TUSC2	Intravenous	I/II	NCT01455389 NCT04486833
Liposome	SGT‐53 (Plasmid with cDNA encoding p53)	Metastatic Pancreatic Adenocarcinoma	p53	Intravenous	II	NCT02340117
Liposome	SGT‐53 (Plasmid with cDNA encoding p53)	Recurrent/Refractory Solid Tumors	p53	Intravenous	II	NCT02354547
Liposome	EphA2 siRNA	Metastatic Solid Tumors	EphA2	Intravenous	I	NCT01591356
Exosome (mesenchymal stromal derived)	KrasG12D siRNA	Metastatic Pancreatic Adenocarcinoma	KrasG12D	Intravenous	I	NCT03608631
Lipoplexes (RNA‐lipoplexes)	Liposomal RNA Vaccination	Unresectable Melanoma	NY‐ESO1, tyrosinase, MAGE‐A3, TPTE	Intravenous	I	NCT02410733
Lipoplexes (SGT‐94)	pDNA	Solid tumors	RB94	Intravenous	I	NCT01517464
Lipoplexes (Atu027)	siRNA	Pancreatic adenocarcinoma	PKN3	Intravenous	I/II	NCT01808638
Lipoplexes (pbi‐shRNA)	pDNA	Metastatic cancer	STMN1	Intratumoral	I	NCT01505153
Poly(α‐l‐lysine) stabilized particle (Hiltonol)	Synthetic dsRNA	Solid tumors, Prostate cancer	TLR3	Intratumoral and intramuscular	II	NCT02423863, NCT06343077
PEG‐PEI‐cholesterol lipopolymer (GEN‐1)	pDNA	Ovarian, fallopian, or peritoneal cancer	IL‐12	Intraperitoneal	I/II	NCT05739981
PEI based nanoparticle (BO‐112)	dsDNA	Solid tumors, melanoma, non‐small lung cancer	TLR3, MDA5, PRK	Intratumoral	I/II	NCT02828098, NCT05265650, NCT04570332
PEI based nanoparticle (SNS01‐T)	siRNA/pDNA	B‐cell lymphoma	eIF5A	Intravenous	I/II	NCT01435720
PEI based nanoparticle (CYL‐02)	pDNA	Pancreatic adenocarcinoma	DCK:UM, SSTR2	Intratumoral	II	NCT02806687
Metabolic
Lipid nanoparticle	Patisiran (Onpattro), siRNA TTR	hereditary transthyretin‐mediated amyloidosis	TTR	Intravenous	—	FDA Approved
Lipid nanoparticle	BMS‐986263 (HSP47 siRNA)	HCV induced cirrhosis	HSP47	Intravenous	II	NCT03420768
Liposome	CFTR plasmid	Cystic Fibrosis	CFTR	Nebulized	II	NCT01621867
Exosome	LDL receptor mRNA	Familial hypercholesterolemia	LDL Receptor	Intravenous	I	NCT05043181

5.1. Metabolic

NA therapy applications have recently pioneered new treatments for incurable neuromuscular diseases that lead to childhood or adult fatality. The FDA has recently approved viral vector‐based treatments for SMA, DMD, and metachromatic leukodystrophy, which are a result of mutations in a single gene, making it well suited as a target for NA therapy.

One siRNA‐based drug with LNPs has been approved by the FDA for treating metabolic diseases. Patisiran (Onpattro) is used as a treatment for polyneuropathy due to hereditary transthyretin amyloidosis (hATTR) (Adams et al. 2018). hATTR is an autosomal dominant disorder due to deposits of abnormally folded transthyretin proteins that deposit throughout the body (Adams et al. 2018). These abnormal systemic deposits result in polyneuropathy and multi‐organ manifestations like cardiomyopathy (Adams et al. 2018). Patisiran is an intravenous infusion of siRNA in large lipid nanoparticles to inhibit liver hepatocyte production of transthyretin. Intravenous injections of BMS‐986263, a LNP containing heat shock protein 47 (HSP47) siRNA underwent a phase II trial in patients with HCV‐induced hepatic fibrosis (“NCT03420768 2022. A Study of Experimental Medication BMS‐986263 in Adults With Advanced Hepatic Fibrosis After Cure of Hepatitis C,”). As HSP47 has been implicated in hepatic fibrosis, siRNA therapy aims to degrade this protein and subsequently treat hepatic fibrosis. 21% of patients in the high‐dose treatment arm (6/28) demonstrated improvement in METAVIR (liver damage) staging compared to 13% of placebo patients (2/15) (Lawitz et al. 2022).

Importantly, lipid nanoparticles have several drawbacks with respect to toxicity related to the excipients in the nanoparticles that may require high doses of anti‐inflammatory medications (Dowdy 2017). The large size of the lipid nanoparticle also limits use to large fenestrated tissues like the liver (Dowdy 2017). The other two FDA‐approved siRNA treatments, Givosiran (Givlaari) and Lumasiran (Oxlumo), which treat acute hepatic porphyria and primary hyperoxaluria type I, respectively, utilize a ligand (GalNAc) conjugated delivery system to target hepatocytes (Balwani et al. 2020; Garrelfs et al. 2021).

Other non‐viral nanoparticles for NA therapy have also been investigated for cystic fibrosis and familial hypercholesterolemia. For familial hypercholesterolemia, which most commonly occurs due to a defect in the LDL receptor, an exosome containing LDL receptor mRNA derived from an LDL receptor virus vector is currently undergoing phase I clinical trials via intravenous injection (“NCT05043181 2021. Exosome‐based Nanoplatform for Ldlr mRNA Delivery in FH (ENDFH),”). No results are currently available on ClinicalTrials.gov (“NCT05043181. Exosome‐based Nanoplatform for Ldlr mRNA Delivery in FH (ENDFH),”).

5.2. Oncologic

Several promising oncological candidates for NA therapy have been investigated in early clinical trials. Namely, delivery of DNA plasmids and nucleic acids (siRNA and mRNA) with lipid nanoparticles holds a promising avenue given a lipid nanoparticle formulation is already FDA approved for drug delivery (Luiz et al. 2022). While other candidates such as exosomes for NA delivery are promising, they are still in limited Phase I clinical trials.

Tumor suppressor candidate 2 (TUSC2) is a tumor suppressor gene inactivated in certain types of malignant lung tumor cells (Rimkus et al. 2017). Given this, restoration of TUSC2 has been employed as an adjunct tool to stimulate apoptotic pathways and immunomodulate signals for patients with lung cancer. The FDA granted a fast‐track designation to lipid DOTAP: cholesterol nanoparticles with tumor suppressor candidate 2 (TUSC2) expression plasmids. The drug (quaratusugene ozeplasmid, Reqorsa) with the LNP delivery system (Oncoprex) was infused intravenously to patients with non‐small cell or small cell lung cancer combination in several phase I/II studies (“NCT01455389 2022. TUSC2‐nanoparticles and Erlotinib in Stage IV Lung Cancer,”; “NCT04486833 2024. Quaratusugene Ozeplasmid (Reqorsa) and Osimertinib in Patients With Advanced Lung Cancer Who Progressed on Osimertinib (Acclaim‐1),”).

Similarly, a cationic liposomal formulation (DOTAP: DOPE) with plasmid encoding wild type human p53 cDNA (SGT‐53) was developed to treat metastatic pancreatic tumors in a phase II trial given p53 is a critical tumor suppressor gene lost in tumors(“NCT02340117 2022. Study of Combined SGT‐53 Plus Gemcitabine/Nab‐Paclitaxel for Metastatic Pancreatic Cancer,”). An interim analysis of stage IV pancreatic adenocarcinoma patients demonstrated median free survival of approximately 7.1 months (n = 11) compared to 3.1 months for approved second‐line therapy, suggesting the clinical benefit of SGT‐53 (Leung et al. 2021). The same liposomal formulation was used in a phase I trial for pediatric patients with refractory or recurrent solid tumors (“NCT02354547 2022. A Study of SGT‐53 in Children With Refractory or Recurrent Solid Tumors”). However, according to ClinicalTrials.gov, the phase I trial has been suspended (“NCT02354547. A Study of SGT‐53 in Children With Refractory or Recurrent Solid Tumors”).

With siRNA, a 1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidylcholine (DOPC) formulation targeting EphA2 is currently undergoing phase I clinical trials in patients with advanced solid tumors at MD Anderson Cancer Center (“NCT01591356 2024. EphA2 siRNA in Treating Patients With Advanced or Recurrent Solid Tumors,”). EphA2 is a receptor tyrosine kinase that is a key regulator in several solid malignancies (Wilson et al. 2021). The DOPC liposomal formulation will be delivered intravenously to determine toxicity profiles and assess any changes in tumor response. Additionally, intravenous infusions of exosomes derived from mesenchymal stromal cells containing KrasG1D siRNA for patients with metastatic pancreatic adenocarcinoma are undergoing phase I clinical trials (“NCT03608631 2024. iExosomes in Treating Participants With Metastatic Pancreas Cancer With KrasG12D Mutation,”).

In advanced unresectable melanoma, an intravenous liposomal RNA vaccination targeting tumor‐associated antigens in melanoma is undergoing testing in an open‐label, multicenter Phase I trial (Lipo‐MERIT) (“NCT02410733 2023. Evaluation of the Safety and Tolerability of i.v. Administration of a Cancer Vaccine in Patients With Advanced Melanoma (Lipo‐MERIT),”). Preliminary results were promising, as exploratory interim analysis demonstrated promising durable responses to therapy in checkpoint‐inhibitor experienced patients (Sahin et al. 2020).

5.3. Neurologic

Drug and NA delivery methods to the central nervous system for neurodegenerative disorders such as Alzheimer's and Parkinson's have traditionally been impaired by the BBB. Nano drug approaches offer a promising alternative given their small size, biocompatibility, and tuning, which allows them to pass through the BBB.

A phase I clinical trial is assessing intravenous infusions of exosomes derived from mesenchymal stromal cells to treat patients with disability after acute ischemic stroke (“NCT03384433 2021. Allogenic Mesenchymal Stem Cell Derived Exosome in Patients With Acute Ischemic Stroke.”). Previous studies have demonstrated the ability of mesenchymal stromal cells derived exosomes to enhance axonal growth in neurons (Waseem et al. 2023; Zhang et al. 2017). Thus, infusions of these exosomes may help patients regain some function after ischemic injury to the CNS (Waseem et al. 2023; Zhang et al. 2017).

Other approaches for NA therapy with respect to neurological diseases are only in the preclinical phase. Banskota et al. engineered a VLP containing Cas9 ribonucleoprotein to obtain therapeutic levels of gene editing in the brain (Banskota et al. 2022). Similarly, Kim et al. developed a modified exosome with T7 peptides to target glioblastoma cells (Kim et al. 2020). Liposomes have been tested in brain tumor models to deliver different chemotherapy agents (5‐FU, methotrexate, paclitaxel) (Hu et al. 2017; Lakkadwala and Singh 2018; Peng et al. 2018). Dendrimers also hold significant promise for NA delivery to the CNS, as several preclinical studies have demonstrated their ability for neural targeting. Dhull et al. developed a 2‐deoxy glucose mixed dendrimer capable of localizing at diseased neurons and with rapid clearance from off‐target tissues (Dhull, Wei, et al. 2024; Dhull, Zhang, et al. 2024).

5.4. Alternative Modes of Administration

Most current FDA approved methods of NA delivery involve systemic intravenous infusion. However, alternative methods of targeted delivery may not only improve efficacy but also reduce systemic side effects and unwanted interactions with other tissues. We discuss current efforts to develop intraocular, intracranial, otologic, and inhalational gene therapy delivery vehicles.

5.4.1. Intraocular Administration

Pathological mutations in any of the 250 genes for retinal function have been mapped to inherited retinal disease (Pontikos et al. 2020). Treatments for retinal diseases currently remain limited, with NA therapy holding huge therapeutic promise. Additionally, the eye is an immune privileged organ with a limited host immune response and requires relatively low quantities of gene vector material (Masli and Vega 2011). To date, only one AAV vector gene therapy, voretigene neparvovec‐rzyl (Luxturna), has been FDA approved for retinal diseases, but many treatments for a variety of retinal diseases are currently in development. Voretigene neparvovec‐rzyl is used for the treatment of Leber congenital amaurosis, a progressive retinal disease, by providing a functional copy of RPE65 in patients with biallelic RPE65 mutation‐related retinal dystrophy (Russell et al. 2017). The medication is delivered via subretinal administration after vitrectomy.

While most ocular NA therapies under clinical trial evaluation are AAV vector based, several nanoparticle‐based approaches are also under evaluation. QR 421a is an RNA antisense oligonucleotide that targets mutations in USH2A exon13 in patients with retinitis pigmentosa, a disease characterized by progressive visual degeneration (Dulla et al. 2021). The medication is delivered via intravitreal injection and was in phase 2/3 trials but terminated in 2024 due to business reasons (Igoe et al. 2024; Pitkänen et al. 2003; Suk et al. 2016). EDIT‐101, a Cas9‐based vector, was used to target CED290‐associated retinal degeneration in a phase 1/2 trial (Hoekstra et al. 2007). Twelve adults and two children underwent subretinal injection of EDIT‐101, with at least 11 participants having improvements in one of the four study efficacy outcomes (Pierce et al. 2024).

5.4.2. Intracranial and Intrathecal Administration

Currently approved FDA NA therapies for neurological disease such as elivaldogene autotemce (Skysona) for cerebral adrenoleukodystrophy are mostly administered via intravenous injection (Keam 2021). By directly targeting central nervous system tissues via intrathecal or intracranial approaches, the risk of systemic toxicity and unwanted targeting of tissues may potentially be minimized. Eladocagene exuparvovec (Kebilidi) is the first FDA approved intracranial gene therapy for direct application to the putamen via stereotactic surgery (Pearson et al. 2020). Eladocagene exuparvovec is an AAV containing cDNA used to treat aromatic L‐amino acid decarboxylase (AADC) deficiency, a disease that leads to a large variety of neurological deficits due to decreased AADC enzyme activity (Pearson et al. 2020).

In spinal muscular atrophy (SMA) patients, as mentioned earlier, onasemnogene abeparvovec‐xioi (Zolgensma) is an FDA‐approved adeno‐associated viral vector therapy that delivers a functional copy of the survival motor neuron 1 (SMN1) gene via intravenous injection. An intrathecal variant (OAV101) is currently undergoing phase 3 clinical trials for SMA type 2 patients (Rayamajhi et al. 2020). For giant axonal neuropathy, a rapidly progressive neurodegenerative disorder from GAN mutations that encode gigaxonin, a phase 1 clinical trial assessed intrathecal administration of scAAV9/JeT‐GAN in 14 patients with early clinical promise (Bharucha‐Goebel et al. 2024; Dhull, Wei, et al. 2024; Dhull, Zhang, et al. 2024).

With nanoparticles, several NA delivery vehicles have also been developed and are undergoing clinical trials. Nusinersen (Spinraza) is an FDA‐approved gene therapy administered intrathecally for all types of SMA. Nusinersen is an antisense oligonucleotide targeting intronic splicing silencer N1 (ISS‐N1) to correct intron 7 splicing (Singh and Singh 2018). For early‐onset Alzheimer's disease, a phase 1 clinical trial is currently evaluating the use of ALN‐PP, a RNAi targeting amyloid precursor protein, that is delivered via intrathecal injection. Similarly, BIIB094, an antisense oligonucleotide targeting LRRK2, a gene associated with Parkinson's disease, underwent phase 1 clinical trials via intrathecal injection (Chen et al. 2016; Taymans et al. 2023).

5.4.3. Inhalation Administration

Inhalational therapies for NA delivery are under extensive clinical development but have yet to receive FDA approval at this time. Cystic fibrosis is an autosomal recessive disease due to mutations in the CFTR gene, which impedes chloride ion transport, resulting in impaired respiratory function, reproductive ability, and pancreatic/GI insufficiency (Shteinberg et al. 2021). Currently available treatments for cystic fibrosis rely on supportive measures, and no FDA‐approved methods for NA therapy exist. A phase IIb trial assessed a catatonic liposome (GL67A) and plasmid DNA with CFTR (pGM169) (Alton et al. 2015). In a cohort of 140 patients, 78 patients received the nebulized liposomal formulation (pGM169/GL67A). There was a statistically significant moderate benefit in forced expiratory volume (FEV₁) compared with placebo at 12 months (Alton et al. 2015). Recently, a phase 1/2 clinical trial assessed the efficacy of inhaled mRNA therapy, MRT5005, aerosolized in lipid nanoparticles in cystic fibrosis (Kawabata et al. 1995; Rowe et al. 2023). The medication was well tolerated in most patients enrolled in the study, with FEV1 remaining stable up to 28 days following last dose administration. In alpha‐1 antitrypsin (AAT) deficiency, lack of protection from AAT leads to liver and lung disease from proteolytic damage (Stoller and Aboussouan 2012). A clinical trial assessed a plasmid cationic liposome delivered intranasally in 5 patients, which demonstrated promise by increasing levels of AAT in the transfected nostril (Brigham et al. 2000).

5.4.4. Inner Ear Administration

NA delivery to the inner ear has significant challenges due to the blood‐labyrinth barrier and physical barriers such as the round window and tympanic membrane (Nyberg et al. 2019). Currently, a phase I/II clinical trial is underway by delivery genetic material with a round window injection approach. An adeno‐associated viral vector (AAVAnc80‐hOTOF) for Otoferlin mediated hearing loss is delivered via trans‐tympanic round window injection to the inner ear (“NCT05821959. Gene Therapy Trial for Otoferlin Gene‐mediated Hearing Loss,”). Nanoparticle approaches to the ear have been limited to preclinical settings, but Sadabad et al. developed elastic liposomes capable of crossing the tympanic membrane and reaching both the middle and inner ear (Kashfi Sadabad et al. 2022). The elastic liposomes may offer a more non‐invasive approach for NA delivery for inner ear applications.

6. Conclusions and Prospects

As of July 2024, over 270 registered gene therapy trials (phases 2 and 3) were listed on ClinicalTrials.gov. As over 10 gene‐based therapies have been approved by the FDA for various genetic conditions, NA therapy is poised to revolutionize the field of medicine. However, almost all FDA‐approved drugs are currently viral vector‐based, and nano‐based delivery methods are only in early stages of clinical translation.

The ideal NA therapy vector should be non‐toxic, non‐immunogenic, and stable without any unintended mutagenesis. Existing viral vector‐based genetic therapies have high transduction efficacy but can suffer from significant immunogenicity and a theoretical risk of mutagenesis. In early clinical trials with gamma‐retroviruses for SCID patients, 2 patients developed leukemia as a side effect of viral vectors due to unintended mutagenesis (Hacein‐Bey‐Abina et al. 2003; Howe et al. 2008). Similarly, nanoparticle delivery methods for NA therapy are in their infancy and no perfectly safe nanoparticles currently exist. The FDA‐approved lipid nanoparticle, Patisiran, has several drawbacks related to the excipients in the nanoparticles that may require high doses of anti‐inflammatory medications and large nanoparticle size that limits use to fenestrated tissues.

Other cationic lipids and polymers can also be associated with cytotoxicity, which limits use (Lv et al. 2006). Further research into the toxicity and safety of these various particles with respect to ideal particle size, chemical groups, and charges will be needed. Additionally, shortcomings in production methods leading to heterogeneity and batch‐to‐batch inconsistency need to be addressed. For example, with exosomes, there is heterogeneous loading efficiency and a lack of standardization with current methods between every synthesized batch.

The cost of NA therapy has also raised questions regarding access to care and health equity. As of July 2024, the most expensive drug in the world is Lenmeldy, a viral vector‐based gene therapy for metachromatic leukodystrophy, costing 4.25 million USD (Mullard 2024). Other FDA gene‐based therapies also cost several million USD per dose. As such, NA therapies are projected to cost approximately 20.4 billion dollars annually in the United States, and true long‐term follow‐up data is lacking at this time (Abuloha et al. 2024). Non‐viral‐based nanoparticles may offer a promising cost‐effective alternative to viral vector therapies if a facile, high‐throughput manufacturing process can be optimized.

Overall, the future of NA therapy with nanoparticles is in its infancy but very bright. The ability to fine‐tune nanoparticles for targeted delivery will allow the development of a nontoxic, non‐immunogenic, and stable vehicle for NA therapy. Additionally, the development of facile, cost‐effective, scalable production methods may help drive down the prices of current NA therapies.

Author Contributions

Joan Castaneda Gonzalez: conceptualization (supporting), investigation (equal), writing – original draft (equal), writing – review and editing (supporting). Ki Wan Park: investigation (supporting), project administration (supporting), writing – original draft (supporting). Dallin Brian Evans: writing – original draft (supporting). Rishi Sharma: conceptualization (supporting), project administration (supporting), writing – original draft (supporting), writing – review and editing (supporting). Om Sahaym: writing – original draft (supporting). Shamila Gopalakrishnan: writing – original draft (supporting). Aqib Iqbal dar: writing – original draft (supporting). Tulio A. Valdez: writing – review and editing (supporting). Anjali Sharma: conceptualization (lead), funding acquisition (lead), investigation (lead), project administration (lead), supervision (lead), writing – original draft (equal), writing – review and editing (lead).

Conflicts of Interest

The authors declare no conflicts of interest.

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Data Availability Statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.