Review on the bioanalysis of non-virus-based gene therapeutics

ABSTRACT

Over the past years, gene therapeutics have held great promise for treating many inherited and acquired diseases. The increasing number of approved gene therapeutics and developing clinical pipelines demonstrate the potential to treat diseases by modifying their genetic blueprints in vivo. Compared with conventional treatments targeting proteins rather than underlying causes, gene therapeutics can achieve enduring or curative effects via gene activation, inhibition, and editing. However, the delivery of DNA/RNA to the target cell to alter the gene expression is a complex process that involves, crossing numerous barriers in both the extracellular and intracellular environment. Generally, the delivery strategies can be divided into viral-based and non-viral-based vectors. This review summarizes various bioanalysis strategies that support the non-virus-based gene therapeutics research, including pharmacokinetics (PK)/toxicokinetics (TK), biodistribution, immunogenicity evaluations for the gene cargo, vector, and possible expressed protein, and highlights the challenges and future perspectives of bioanalysis strategies in non-virus-based gene therapeutics. This review may provide new insights and directions for the development of emerging bioanalytical methods, offering technical support and a research foundation for innovative gene therapy treatments.

1. Introduction

Genes play a role, to a greater or lesser extent, in nearly all diseases. Researchers have identified more than 3400 genes related to the disease occurrence and development [1]. Gene therapy, a groundbreaking technique, has the potential to resolve a diverse array of medical conditions, encompassing chronic infection (such as HIV/HBV), neurodegenerative disease, cardiovascular disease, cancer, and other rare diseases [2]. Gene therapy as defined by the Food and Drug Administration (FDA), is a technique that modifies a person’s genes to treat or cure disease [3]. Over the past years, the definition of gene therapeutics has expanded, to include nucleic acid modalities that manipulate the gene expression specifically in cells to treat disease. Generally, the modalities can be categorized into three parts: gene deletion/modification to achieve a gain or loss of function like CRISPR/Cas9, enhancement of particular gene expression by DNA plasmid (pDNA), minicircle DNA (mcDNA), messenger RNA (mRNA), and self-amplifying RNA (saRNA), the post-transcriptional modulation involving in small interfering RNA (siRNA), antisense oligonucleotides (ASO), and microRNA (miRNA) [4–6].

DNA/RNA nucleic acids are very vulnerable in their naked form, and various enzymes in biofluids can degrade them. This may trigger undesired immune responses and result in the loss of function and quick elimination. Consequently, this has led to the need for suitable vectors that can: firstly, protect genetic material from degradation; secondly, deliver genetic product to the target site while crossing multiple extracellular and intracellular barriers; and thirdly, mitigate unwanted immune responses [7,8].

Gene therapeutic vectors are generally divided into two main categories: the viral and non-viral based vectors. Both types have their own unique advantages and disadvantages. Virus vectors, including retroviruses/lentiviruses, adenoviral vectors, adeno-associated viruses, and other viruses, demonstrate high transfection efficiency and stable gene expression. However, they also have limitations including toxicity, immunogenicity, carcinogenicity, high cost [9,10]. In contrast, non-viral vectors possess many advantages, such as low immunogenicity, biodegradability, easy synthesis, cost-effectiveness in production, and flexibility in the size of encapsulated gene materials [11,12].

Until March 2023, there are total of ~3900 gene therapeutics clinical trials worldwide (according to Gene Therapy Clinical Trial Worldwide database provided by the Journal of Gene Medicine) [13]. Currently, gene therapies in clinical trials still mostly use viral vector systems, while non-viral vectors are less used. The non-viral based gene therapy applications can roughly be divided into two categories: as a vaccine to prevent certain diseases or as a therapeutic agent to replace, delete, or modify the expression of certain protein or to alter its function. Besides the proven effectiveness of mRNA vaccines against the COVID-19 pandemic, there are many ongoing mRNA/DNA vaccines pipelines, including vaccines against infectious diseases such as influenza (Influenza), Respiratory syncytial virus (RSV), tuberculosis (TB), human papilloma virus (HPV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and bacterial infections. Furthermore, cancer vaccines also play a significant role in the realm of cancer immunotherapy, mainly targeting melanoma, ovarian cancer, urothelial cancer, and lymphoma [14–16]. Act as therapeutic agents to treat particular diseases, there are many ongoing pipelines. These include the inhibition of specific protein expression using RNAi agents (siRNA, miRNA) and ASO [17–20], protein supplement or replacement using mRNA or DNA [21,22], and the deletion/excision of intragenic DNA by CRISPR/Cas9 components [23,24].

Currently, there are 10 approved non-viral based gene therapeutics worldwide (until Oct. 2024), including five siRNA therapeutics utilize GalNAc conjugate technology, three lipid nanoparticle (LNP) based, and two plasmid DNA-based gene therapeutics. Patisiran, the world’s first LNP-siRNA drug was approved by FDA in October 2018 [25], and in 2020 the Pfizer-BioNTech BNT162b2 and the Moderna mRNA-1273 LNP-mRNA vaccines were emergently approved against SARS-CoV-2 [26,27]. There are two plasmid-based gene therapeutics. The Neovasculgen encoding vascular endothelial growth factor (VEGF) was approved in the Russian market in 2012 for treating peripheral artery disease [28]. The Collategene encoding hepatocyte growth factor (HGF) was approved in Japan market in 2019 for the improvement of ulcers in patients suffering from peripheral artery disease [29]. To be noticed, the GalNAc conjugate can consider as a kind of vector which could facilitate the liver tissue targeting. There are five GalNAc conjugated siRNA therapeutics, including Givosiran, Lumasiran, Inclisiran, Vutrisiran and Nedosiran [30].

Based on past knowledge, the system drug concentration is the key driver of its efficacy and toxicity for conventional small or large molecular drugs. However, the systemic circulation half-life of gene therapeutics is considerably much shorter than the tissue half-life. The tissue exposure mainly determines its pharmacological effects and toxicity [31,32]. So, it is necessary to know the PK/TK, biodistribution, and immunogenicity for each component of the non-virus based gene therapeutics, including the carrier, gene cargos, and potential protein products. Biodistribution assessments are required components of investigational and final drug or biological approval applications by the FDA, the European Medicines Agency (EMA), the Japan Pharmaceutical and Medical Devices Agency (PMDA), and other regulatory authorities [33]. Some polymer-based nanocarriers may be challenging to degrade in vivo, such as polyethyleneimine (PEI) vectors [34]. These polymers’ metabolism or excretion pathways need to be clarified to assess their potential toxicity and body tolerability.

In the paragraph below, we will first discuss the basic structure of non-viral based gene therapeutics. Secondly, we will dive into the general bioanalysis strategies for the quantification/semi-quantification of gene cargo, vector carriers, and potential protein products in systemic circulation and tissues, and the immunogenicity consideration and assessment for these components. Finally, we will summarize the bioanalysis-related perspective concerning the non-viral based gene therapeutics.

2. Basic structure of non-viral based gene therapeutics

The non-viral based gene therapeutics consist of the gene cargo and its vectors. The gene cargo varies from the short oligos like ASO [35], siRNA [36,37], miRNA [38] to the long DNA [39], mRNA [40], saRNA [41] and CRISPR/Cas9 genome-editing components [42].

The delivered gene cargo mechanism includes the occupancy-mediated degradation or occupancy-only mediated gene regulation by ASO, the RNA interference-mediated gene regulation by siRNA or miRNA, the protein translation by foreign mRNA and the transcription and translation by external DNA, the gene deletion/modification by CRISPR/Cas9 or similar systems [43–45]. To be noticed, the DNA or CRISPR/Cas9 components need to reach the nuclear to perform its function. The nuclear entry requires either the disintegration of the nuclear envelope during the process of cell division or active transport through the nuclear pore complex (NPC) [46,47] (Figure 1).

Figure 1.

The schematic diagram of the general mechanism of non-virus based gene therapeutics. The non-viral based gene therapeutics consist of the gene cargo and its vectors.

The non-viral vectors vary in the chemical component and physical properties. Although there are many different types, their design usually meets the following functional requirements. First, it can protect gene cargo from the degradation by endogenous nucleases. Second, it can protect the gene cargo from unwanted endocytosis by the reticuloendothelial system and macrophages. Third, it neutralizes the negative charge of the gene cargo to facilitate cell uptake. And finally, it facilitates the endosomal escape. The most extensively researched non-viral vectors are mainly polymers, lipid-based vectors, and inorganic nanoparticles [4,48]. Most of the vectors are cationic, enabling them to interact with DNA/RNA, which possess negative charges. The vector-gene complex is designed to bind with the negatively charged molecules on the cellular membrane, internalize to the cell, escape from endosome/lysosome degradation, and deliver the transgene to the cytoplasm and nucleus.

Various polymers such as dendrimers like poly propylene imine (PPI) and polyamidoamine (PAMAM); polylactic acid (PLA); polyethylenimine (PEI); and chitosan (CS) have been widely used as delivery systems [49,50]. Lipid-based vectors are spherical vesicles composed of one or more bilayers of lipids surrounding an aqueous core. The lipid nanoparticle (LNP) is currently the most promising lipid-based vector candidate for gene delivery. LNP mRNA vaccines have achieved great success in quickly responding to the COVID-19 pandemic and with effectiveness in disease prevention [51]. The LNP comprises ionizable cationic lipids, cholesterol, phospholipids, and polyethylene glycol (PEG)-lipids. To achieve better tissue or particular cell group specificity, the next generation “smart” LNP are being modified to have ligand targeting or stimulus responding capabilities [52,53]. The inorganic nanoparticles include metallic nanoparticles, quantum dots, carbon nanotubes, and silica-based nanoparticles. They can be engineered to have particular structures [54,55]. The physical properties and electric charge entities render them suitable candidates for use as vectors in gene delivery application.

3. Bioanalysis of non-viral based gene therapeutics

Due to the complex nature of the non-viral-based gene therapeutics. In order to fully understand each component’s PK and biodistribution pattern, the corresponding bioanalysis can be roughly divided into four parts, including the bioanalysis of the gene cargo, the bioanalysis of vector or its representative component, the bioanalysis of the target or expressed protein, and the immunogenicity evaluation. The detailed contents for each bioanalysis part are introduced as follows.

3.1. Bioanalysis of the gene cargo

The suitable bioanalysis strategies vary depending on the size/length of the gene cargo. Based on the size, the gene cargo can roughly divide into oligonucleotides (OGNs) and long nucleic acid. OGNs generally includes siRNA, miRNA, ASO, and gRNA which are typically less than 50 base pairs (bp) in length [56]. Long nucleic acids comprise the mRNA or DNA, which range from hundreds to thousands of bases.

3.1.1. Bioanalysis of short OGNs

The choice of proper OGNs bioanalysis platform varies depending on the assay requirements, such as sensitivity, ability to discriminate metabolites, multiplex, throughputs, and quantification or semi-quantification. Strategies including low- or high-resolution liquid-chromatography mass-spectrometry (LC/MS), hybridization ligand binding assay (LBA), hybrid LBA-LC/MS, and polymerase chain reaction (PCR) are mainly employed for the OGNs bioanalysis. In addition, various analytical techniques also have been employed for the OGNs bioanalysis [57], quantitative whole-body autoradiography (QWBA) [58,59], capillary gel electrophoresis (CGE) [60], LC with ultraviolet (UV) or fluorescence detection [61], matrix-assisted laser-induced desorption/ionization mass spectrometry (MALDI) [62]. These analytical strategies have their unique application scenarios. For example, MALDI-imaging can evaluate the biodistribution of OGNs and their metabolites in a delicate spatial manner [62,63]. This review mainly introduces the LC/MS, hybridization LBA, hybrid LBA-LC/MS, and PCR platform for OGNs absolute quantifications, highlighting their individual unique advantages, disadvantages, and application scenarios (Figure 2).

Figure 2.

The schematic diagram of different bioanalysis strategies for OGN. The advantages and disadvantages of the OGN bioanalysis on the LC/MS, LBA, hybrid LBA-LC/MS and PCR platforms are listed below.

3.1.1.1. LC/MS-based approach

The LC/MS based approach is widely used for the OGNs bioanalysis, due to its unrivaled specificity and wide dynamic range. Compared with LBA and qPCR platforms, it is excellent in its ability to discriminate the metabolites from the parent OGNs and monitor them in parallel [64]. The proper sample preparation and extraction steps are critical for the OGNs bioanalysis when using LC/MS platforms to ensure the recovery and assay robustness, due to the plasma protein binding, unspecific binding with positively charged consumables, and stability issues. The common strategies for sample preparation including, protein precipitation, enzyme digestion, liquid-liquid extraction (LLE), solid phase extraction (SPE), and a combination of these strategies above [65]. The LC/MS assay is generally preferred if the assay sensitivity (~5–25 ng/mL for plasma) is adequate for the study. Also, multiple OGNs (and metabolites) can be quantified and monitored simultaneously in one assay. To be noticed using nano/microflow LC (increasing the ionization efficiency and chromatograph resolution) or a sequential sample extraction strategy (improving recovery and signal to noise ratio) can further improve the sensitivity of OGN bioanalysis but will sacrifice the throughput of the assay [66–69].

3.1.1.2. LBA-based approach

The LBA based approach, such as hybridization enzyme linked immunosorbent assay (ELISA) or meso scale discovery (MSD) can also be utilized for oligonucleotide quantification. LBA methods have the advantage of high sensitivity and robustness while requiring minimal sample preparation steps, which could increase the assay’s throughput. However, compared with LC/MS platforms, the LBA methods are limited by their narrow dynamic range and have less ability to discriminate OGNs intact form with its shorter metabolites [70,71]. There are many different hybridization-based assay formats for OGNs bioanalysis, including the sandwich-based assay [72], competitive hybridization assay [73], one-step hybridization [74], two-step hybridization-ligation assay [74], and dual ligation hybridization assay [75]. The ligation-based approach could significantly reduce the 3’ and 5’ metabolite interference. The two-step hybridization-ligation assay could reduce the cross-reactivity of 3’ N-1 to 8.3% [74]. The dual ligation hybridization assay could reduce the cross-reactivity of 3’ N-1 to 4%, and 5’ N-1 to an undetectable level [75]. However, the ligation-based approach will use multiple primers/probes and enzymes in the assay. Utilizing such a sophisticated system for the routine or regulated bioanalysis of oligonucleotides will present significant challenges.

3.1.1.3. Hybrid LBA LC-MS based approach

In recent years, a novel hybrid LBA LC-MS methodology was developed for the quantification of oligonucleotides in biological samples [76–78]. The LBA-LC/MS hybrid approach harnesses the strengths of both LC/MS and LBA platforms with great sensitivity while maintaining high specificity. This process employs a capture probe (typically a complementary DNA sequence) to specifically hybridize to the OGNs to achieve highly efficient sample purification. Although the capture process may still extract the shorter metabolites, the LC-MS analysis can differentiate them from the full-length OGNs and can be quantified and monitored simultaneously in one assay. Hybrid LBA LC-MS has demonstrated its capability to analyze the ASO [77,79,80], siRNA [81] and miRNA [82]. Its sensitivity and specificity make it an invaluable tool in the field of OGN research and analysis.

3.1.1.4. PCR-based approach

The PCR is an enzyme-based assay widely used in molecular biology research and diagnostics. This method involves the amplification of template DNA or reverse transcribed (RT) complementary DNA (from RNA) through a series of thermal cycles (typically ranging from 40 to 45 cycles), using short complementary primer probes. Two primary detection systems are employed to quantify the amplified DNA: SYBR green dye, which intercalates into double-stranded DNA products, and TaqMan fluorescent probes, which release a signaling group upon cleavage during the synthesis of daughter strands. In the quantitative PCR (qPCR) methodology, the quantity of labeled products is measured in real time following each cycle of amplification. For the digital PCR (dPCR) approach, the whole mixture is split into thousands of small compartments of reaction and output is generated from the reading of each ‘compartment’ after completion of 40–45 cycles. The dPCR approach eliminates the necessity of a standard curve, relying instead on Poisson distribution statistics to determine the absolute quantity of template copies [83]. Compared with the qPCR approach, the dPCR approach has better sensitivity, precision, and tolerance to PCR inhibitors, but also has shorter dynamic range, less throughput (up to 96-well for dPCR compared with 384-well for qPCR), and relatively higher cost [84,85].

The PCR platform serves as a highly effective and sensitive technique for OGNs quantifications. With proper primer/probe design, the sensitivity of the qPCR method could be achieved to attomole level [86]. However, the short length of OGNs often presents challenges in developing appropriate primer pair and a probe oligonucleotide. To solve this issue, modified methodologies have been developed to ‘extend’ the length of target OGNs before performing qPCR. These methodologies include the stem-loop RT-qPCR [87,88], two-tailed RT-qPCR [89], primer-extension qPCR [90], ligation-based qPCR [91,92]. Usually, the PCR-based assay is hard to distinguish the intact OGNs from their shorter metabolites. Due to the intrinsic variability associated with the PCR-based method, the acceptance criteria for precision and accuracy were generally much larger than the conventional LC/MS or LBA approach (±30%/±50% compared with ± 15%/±20% or ± 20%/±25%) [93]. dPCR is also applied for the absolute quantification of OGNs [94]. It employs a similar assay primer/probe design as described above for conventional qPCR, but counts the total number of individual target molecules in a digital format for final quantitation. The PCR-based method is ideal for the assay requiring high sensitivity, such as the quantification of RNA-induced silencing complex (RISC) loading siRNA [95].

3.1.2. Bioanalysis of long nucleic acid

For longer nucleic acids like mRNA or DNA, the whole sequence often spans thousands of nucleotides. Generally, the branched DNA (bDNA), qPCR, and dPCR-based platforms are primarily used for mRNA/DNA quantification [96]. Because of its extremely high molecular weight and poor ionization efficiency, it is almost impossible to obtain intact quantification of long nucleic acids through LC/MS. However, the LC/MS method could be a powerful tool to characterize mRNA, including the nuclease-based sequence mapping [97], RNA post-transcriptional modification (PTM) evaluation [98], and integrity evaluation (poly-A tail and cap) [99]. The acid hydrolysis isotope dilution LC/MS approach could be used to quantify pure or matrix-based mRNA in LNP [100]. There are also other technologies available to quantify or semi-quantify mRNA/DNA. For example, the fluorescence in situ hybridization (FISH) method is extensively employed for mRNA visualization and semi-quantification on fixed samples [101,102]. The RNAscope/ViewRNA techniques use a similar assay principle with bDNA, and can provide both semi-quantitative and tissue localization data for mRNA vaccines [103]. And the Picogreen/Ribogreen fluorometric assay is mostly used to assess the encapsulation of nucleic acids, which can effectively assess the stability of drugs under different environmental conditions. The degree of encapsulation directly determines the efficacy of the drug in vivo, therefore, the encapsulation rate is one of the key quality attributes that need to be evaluated for non-viral vector drugs [104]. The following section will primarily explore methodologies for absolute quantification of long mRNA and DNA utilizing the bDNA, qPCR, and dPCR-based approaches (Figure 3).

Figure 3.

The schematic diagram of different bioanalysis strategies for long nucleic acids, mRNA as an example. The advantages and disadvantages of the mRNA bioanalysis on the bDNA and PCR (qPCR/dPCR) platforms are listed below.

3.1.2.1. Branched DNA (bDNA) approach

Academic laboratories have used the bDNA to quantify long nucleic acids for years. It has been widely used for the quantification of viral load for HIV, HCV, and HBV infections [105]. The assay employs a hybridization method involving customized capture, block, and Z-extender probes, which target a significant portion of the target mRNA sequence. The amplification probe could specifically recognize the dual Z-extender probes and amplify the signals using detection reagents. The bDNA assay is adaptable for use in either a tailored plate format or beads-based format. Based on a similar principle, the RNAscope or similar assay offers a powerful tool for the in-situ hybridization (ISH) of target mRNA/DNA within intact cells or tissue slices [106,107]. It has been wildly used to detect viral infection and analyze the gene expression within tissue specimens [108], and has recently been used to evaluate the tissue distribution of a COVID-19 mRNA vaccine (mRNA-1647) in rat [109] and an mRNA influenza vaccine that encoding hemagglutinin in mouse [110]. In addition, ISH co-staining with immunohistochemistry (IHC) could detect mRNA and its expressed protein on the same tissue slide [111]. The bDNA approach has also been used for the LNP-encapsulated mRNA quantification [103]. For some highly collagenous or hard tissues, the alternative extraction buffer (containing β-mercaptoethanol), liquid nitrogen grinding or additional phenol extraction purification steps could help increase the mRNA extraction efficiency from its LNP structure [112].

The advantage of bDNA approach over PCR-based platforms is its simplified sample preparation steps, eliminating the requirement for prior extraction and purification of mRNA from the sample before analysis [112]. The assay is based on a direct measurement of long nucleic acid itself. For the PCR based assay, the reverse transcription (for mRNA) and amplification step may be more sensitive to the small turbulence of the assay condition. The bDNA approach generally has better accuracy and precision and could meet the LBA acceptance criteria for the regulated environment [96,113]. However, the bDNA assay also has some disadvantages. The signal responses of bDNA assay may vary in different matrices [114]. For the mRNA rich tissues, such as the liver or kidney, the bDNA assay’s sensitivity is not as good as that of less mRNA containing tissues, for example the muscle or heart. And the assay may need more dilution to avoid the selectivity and matrix effect for specific tissues. Based on our experience, the bDNA assay is remarkably suited for the circulation PK study but will need more optimization for the tissue distribution study. Another limitation is that the bDNA approach cannot distinguish the intact or partially degraded mRNA/DNA. To ensure the sensitivity of the assay (signal amplification), multiple Z-extender probes are designed to cover a large region of the mRNA/DNA, allowing the detection of the target sequence even if some parts of the structure are lost.

3.1.2.2. PCR-based approach

The qPCR and dPCR platforms are mainly used for long mRNA/DNA bioanalysis. Generally, the bioanalysis for tissue distribution will require very high sensitivity to fully understand the risk for genome editing for the gene therapeutics which could have the potentials, like CRISPR or pDNA based therapeutics. The FDA guidance recommends that PCR assays for biodistribution have a demonstrated LLOQ of ≤50 copies of target vector per 1 μg of host genome DNA (gDNA) with 95% confidence [115,116]. The dPCR has advantages in sensitivity and could be more suitable for this type of assay. The PCR-based approach has been widely used for PK and biodistribution study for gene therapeutics. The oncoprotein encoding mRNA for a cancer vaccine was quantified by the qPCR method [117]. The amount of COVID-19 vaccine mRNA (mRNA-1273 or BNT162b2) was measured by qPCR from human plasma [118], and autopsy tissues [119]. And to evaluate the mRNA-1273 and BNT162b2 vaccine mRNA’s capability to cross the blood-breast barrier and blood-placental barrier, the mRNA concentration in breast milk and placental explants were quantified by qPCR [120,121] and dPCR [122] methods. The biodistribution of several self-amplifying mRNA (saRNA) vaccines was evaluated by the qPCR platform in mice [123,124] and rats [125,126]. Similarly, the biodistribution of some plasmid DNA based therapeutics were also evaluated by qPCR method [127,128].

Compared to the bDNA method, the PCR method has many advantages. It offers better sensitivity (dPCR is even more sensitive), which could be beneficial for the risk evaluation of heritable genome edition or the potential of crossing the blood-breast, blood-placental, and blood-brain barriers. Also, the PCR-based assay provides big flexibility in customizing assays to align with the study’s requirements. With the properly designed primer sets, the PCR-based approach can help to understand the integrity of certain regions of long mRNA/DNA. The disadvantages of the PCR-based approach are that it generally needs to extract the nucleic acid before quantification, which may introduce more variance and recovery issues. The reverse transcriptase and polymerase activity may be interfered by several factors or inhibitors. For the PCR based assay, multiple sample extraction and enzymatic reaction steps could accumulate large intrinsic variance for absolute quantification. And most importantly, there is no official guidance yet for the PCR based assay validation and sample analysis, the PCR based bioanalysis in preclinical and clinical stages are still mainly a fit for purpose design.

3.2. Bioanalysis of the non-viral based gene vector

For the non-viral based gene therapeutics, some of the vectors have been well studied. Their safety and ADME characteristics have been fully researched. Many of their components are safe and demonstrated good biocompatibility to human, such as the cholesterol, phospholipids from LNP structures, and synthetic polymers like PEG/PLA which have been approved by FDA for human use over decades. Generally, it is not necessary to monitor the PK and biodistribution for these vectors or their components. But for the vector components that are new or their ADME traits not fully studied, it is necessary to know their biodistribution, metabolism and clearance pathways which could be related to the potential toxicity due to the tissue accumulation.

The bioanalysis of non-viral vectors presents significantly greater challenges compared to the analysis of conventional small molecular drugs. This is primarily due to the large size, heterogeneity, and complexity of several components. The polymer-based vectors could vary in the monomer numbers and chain structure (linear or branch). The lipid-based vector is a mixture of ingredients, for example, the LNP typically consists of four major components: phospholipids, cholesterol, cationic/ionizable lipids, and PEGylated lipids. The inorganic nanoparticles could vary in physical properties and are usually in nanometer size. The selection of an appropriate bioanalysis method for measurement often depends on the vector’s physical properties and chemical composition in a fit-for-purpose design. Multiple strategies can be used to evaluate PK and biodistribution for the non-viral based gene vector, including the LC/MS, inductively coupled plasma (ICP)-MS, LBA, radiolabeling, and fluorescence labeling. If it is hard to monitor the vectors as a whole, sometimes we can monitor the surrogate ingredient to represent the PK/biodistribution profile of the whole vectors.

LC/MS-based approach is widely used for the bioanalysis of polymers like PEG [129,130], PLA [131], hyaluronan (HA) [132], and chitosan [133]. Moreover, this approach is also employed for the bioanalysis of the surrogate lipid components for LNP, such as the PEGylated lipids [134], ionizable lipid SM-86 [135], SM-102 [136], and MC3 [137]. The LBA-based approach can also be used to quantify PEG vectors using plate or cell-based ELISA [138–140]. It enables the tracing of vectors behavior in living organisms, providing a quantitative assessment of their biodistribution, drug targeting, and clearance by labeling nanoparticles with radionuclides. For pre-clinical study, the quantitative whole-body autoradiography (QWBA) is employed to assess the distribution of radiolabeled vectors in rat tissues for Patisiran, BNT162b2, and mRNA-1273 [141]. Fluorescence microscopy serves as an effective technique for monitoring the distribution of nanoparticles at both cellular and tissue levels [142]. To be noticed, the autofluorescence difference of native tissues may interfere with the detection. For the metal based inorganic nanoparticles, the ICP-MS and electron microscopy can also be employed for the PK and biodistribution evaluations [143–145]. For the non-biodegradable polymers or inorganic nanoparticles, it is necessary to do the mass balance study to determine the excretion routes and identify the metabolites, the LC-MS, radiolabeling, and fluorescence labeling techniques can be applied [146].

3.3. Bioanalysis of the expressed protein

For gene therapeutics expressed protein, the bioanalysis strategies are similar to those of protein-based therapeutics. Multiple assay platforms can be used for the PK and biodistribution evaluation of the gene therapeutics expressed protein, including LBA, LC/MS, hybrid LBA-LC/MS, western-blot (WB), and immunohistochemistry (IHC). Below will mainly introduce the platforms for the absolute quantification (Figure 4). Each strategy will have its own advantages/disadvantages and application scenarios. For the LBA assay, there are multiple commonly used platforms can be applied, which are all based on a similar immunoassay principle but various in the signal-generation mechanisms. For example, the ELISA assay based on the enzymatic developed color [147], the MSD or similar assay based on the electro-chemiluminescence (ECL) developed luminescence [148], the Gyrolab/Ella/Luminax/single molecule counting (SMC)/single molecule array (SIMOA) or similar assay based on the fluorescent developed light [149–153], SIMOA planar array assay or similar assay based on the chemiluminescence (CL) developed light [154]. The choose of proper assay platforms depends on the requirement of assay sensitivity, dynamic range, throughput and turnaround time. Several mRNA-encoded vaccine antigens [155], therapeutic antibodies [156], and protein replacement [157] were determined by ELISA assay.

Figure 4.

The schematic diagram of the LBA based and LC/MS based bioanalysis strategies for the target or expressed protein.

Recently, the LC/MS-based approaches have gained significant traction in the bioanalysis of protein-based therapeutics. Generally, protein quantification is often performed by first digesting the target proteins and then analyzing one or more corresponding surrogate peptides. To further improve the sensitivity of the assay, the high-abundant protein depletion, and immunocapture enrichment (hybrid LBA-LC/MS) approaches can be applied [158]. Compared with the LBA assay, the LC/MS based assay has advantages in selectivity, domain mapping, and multiplexing. Notably for the LBA based assay, it could be a challenge to screen a proper antibody pair to discriminate the gene therapeutic expressed protein with its similar endogenous protein. However, the LC/MS or hybrid LBA-LC/MS based surrogate peptide approach could be a powerful tool to distinguish these two based on the unique peptides.

3.4. Immunogenicity evaluation

The immunogenicity evaluation of the non-viral based gene therapeutics shall consider at least three aspects: the immunogenicity against the gene cargo (e.g., against DNA/RNA transgenes), the immunogenicity against the gene vector (e.g., liposome or polymers), and the immunogenicity against the expressed protein (e.g., mRNA/DNA based protein replacement therapy). To be noticed, the preexisting anti-drug antibody (ADA) may also need to be monitored. For example, if the gene vector contains PEG, the preexisting anti-PEG antibody may influence the PK and efficacy of the gene therapeutics, thus need to be investigated the impact on PK/PD [159].

The bioanalysis method for anti-RNA/DNA, anti-vector, and anti-expressed protein antibodies should be developed and validated separately. It is essential to employ risk-based approach to evaluate the potential immunogenicity associated with non-viral based gene therapeutics. In the context of LNP-mRNA therapeutics, there appears to be a reduced necessity for anti-mRNA immunogenicity assays. Current evidence suggests that there is no substantial triggering of immunogenic responses directed against mRNA in the absence of autoimmune conditions [160]. The risk of anti-RNA ADA is considered low. However, the anti-expressed protein ADA shall be monitored for mRNA-mediated protein replacement therapy. The immunogenicity of LNP vector can be considered as low. Still, the PEG component on the LNP vector could induce anti-PEG ADA, which may impact the safety and efficacy of LNP-mRNA vaccine [161]. The measurement of anti-RNA/DNA ADA and anti-vector ADA can be conducted using a plate-based ADA direct assay. To facilitate the plate-based ADA assay, test articles can be coated on different types of microtiter plates using various strategies. For example, the mRNA can be coated on the mRNA Catcher™ plate, the DNA and OGNs can be coated on the polystyrene microtiter plates using the DNA coating solution [162], and the PEG components can be coated on the hydrophilic surface microtiter plates [163]. The anti-expressed protein ADA generally be measured using a classic ADA bridge assay. Besides the evaluation of humoral immunogenicity, the cellular Immunogenicity of specific T cells can be monitored by ELISpot [164] and intracellular cytokine staining (ICS) [165] methods.

4. Other bioanalysis-related perspectives

4.1. Biomarker perspectives

The biomarker related bioanalysis of non-viral based gene therapeutics is always a fit for purpose design, which is generally associated with the assessment of the drug’s pharmacodynamics (efficacy) and toxicity (safety). The selection and evaluation of biomarkers vary depending on the design, structure, and mechanism of action (MOA) of the gene therapeutics. Biomarkers can provide critical insights into a gene therapy’s effects and mechanisms. For example, it is conductive to the patient selection, which identifies the right patient population for a specific gene therapy based on gene sequencing analysis. The proper endpoint biomarkers selection could help to determine whether a drug is effective or not. The immune monitoring involves evaluating the T-cell response to either the vector or the target protein in addition to assessing the inherent and acquired immune response. From a drug development perspective, the biomarker related bioanalysis may require different strategies and flexibility. For example, during the discovery stage, the biomarker assay may generate data demonstrating whether the drug is efficacious or not, and the exploratory bioanalysis strategies could apply even semi-quantitatively. When entering the investigational new drug (IND) or clinical phase, the assay may require a validation/qualification from an exploratory setting into a regulated environment to further prove the assay’s precision, accuracy, selectivity, robustness, and system suitability. In recent years, a multitude of omics technologies, including DNA-seq/RNA-seq, proteomics, metabolomic have become integral components of clinical research in many diseases. The Human Cell Atlas represents the first substantial initiative to integrate these omics technologies, paving the way for the identification of alternative biological biomarkers that can enhance new drug development and efficacy analysis. This groundbreaking approach promises to significantly improve our understanding of immune responses and facilitate the discovery of more targeted therapeutic strategies [166].

4.2. Sample preparation perspectives

Many factors related to the sample preparation could affect the quantitative accuracy, especially for the tissue distribution study, for example, the cross-contamination during the tissue sampling, the sampling region bias, and the stability of gene cargo. For gene therapeutics, bioanalysis methods are generally very sensitive, and many reasons could cause false positive results of biodistribution. Contamination may come from the dissection or homogenization tools are not thoroughly cleaned across samples, or from tiny liquid droplets or aerosols generated during the process. To overcome the challenge, it is recommended to employ the tissue sampling order strategies (from least to highest expected presence of test article), and use disposable consumables [115]. For PCR assays with high sensitivity, using the PCR workstation/cabinet that has separate areas, including the areas for master mix preparation, pre-PCR, amplification, and post-PCR, could help to avoid cross-contamination [167].

Suppose only a small section of a large organ is analyzed, accurate results may not be achieved due to the potential local bias for the target within the organ, especially in large animals. Extracting the whole organ could solve the issue but may be costly due to the need for large amounts of enzyme, lysis buffer, and the need for reaction time optimizations. Sampling from the liquid-nitrogen frozen crushed and mixed organ could avoid the region bias issue. Additionally, it is crucial to take into account the stability of the analyte, especially for the RNA-based gene cargo. The gene cargo may be very vulnerable to the endogenous endonucleases released during tissue sample preparation, which could degrade the unprotected gene cargo. Several strategies could be applied to avoid the degradation, including using phenol/chloroform (TRIzol) lysis buffer to denature the enzyme [168], pretreating the tissue with RNAlater [169], or employing a PAXgene tube for peripheral blood collection [170]. The DNase/RNase free water and consumables are also recommended for use. With careful attention to these considerations during sample preparation, researchers can ensure improved quantitative accuracy and minimize potential sources of error in tissue distribution studies.

4.3. Regulatory perspectives

The bioanalysis method should be considered to be qualified or validated to ensure accurate and robust quantification. For gene therapeutics, several regulatory documents have highlighted the employment of unconventional bioanalytical techniques to evaluate the safety of clinical endpoints [171]. However, there is minimal detail on acceptance criteria and which validation items should be performed. Many bioanalytical labs adhere to the method validation guidance for ligand binding or chromatography-based assay, as provided by FDA, EMA, and International Council for Harmonization (ICH) when a similar assay principle applies [172]. For example, the hybridization assay for OGN and bDNA assay for mRNA/DNA could reference the LBA regulatory guidance, the LC/MS assay for gene vectors like cationic lipid or PEG lipid could reference the chromatography regulatory guidance. The regulatory perspectives on the novel assay platform like PCR and hybrid LBA-LC/MS will be introduced below.

The PCR-based approach is referred for DNA/RNA quantification by guidelines from EMA, ICH, and the International Pharmaceutical Regulators Programme (IPRP) [141]. Although the guidance recommends PCR approaches for the mRNA/DNA bioanalysis, the exact method validation items and acceptance criteria have not yet been defined. Several white pages or publications offer standardization suggestions for qPCR/dPCR based bioanalysis [113,173–175]. The Global CRO Council in Bioanalysis (GCC), comprising contributors from 26 distinct contract research organizations, presents a unified perspective on the essential validation parameters for applicable qPCR/dPCR assays [176]. The hybrid LBA/LCMS approach for biotherapeutics has been used for more than ten years, with many published papers [177–179]. However, there are limited IND and biologics license applications (BLA) submissions using this approach to date. Due to the nature of using LBA techniques, the acceptance criteria should be established based on the data obtained from the validation process, and broader acceptance criteria for precision and accuracy of LC/MS platform (within 15%/20% LLOQ and ULOQ) may be considered [179,180].

As more gene therapeutics development pipelines emerge, bioanalysis scientists will face increasing challenges for assay platform selection, method design, and validation. New assay platforms like PCR and hybrid LBA-LC/MS offer advantages in bioanalysis but will also present method validation challenges from the regulatory perspective. Generally, it is essential to implement fit-for-purpose approaches for method validation, including evaluation of accuracy and precision, specificity, selectivity, and stability to confirm the method’s appropriateness and maintain data integrity. Additionally, sponsors should always be ready to address any supplementary inquiries from regulatory agencies who are conducting their due diligence, particularly when novel technologies are introduced.

4.4. Future perspective

As research in gene therapy continues to advance, non-viral based gene therapy is emerging as a safe and effective alternative that garners increasing attention. Currently, technological advancements in analysis platforms have significantly contributed to the characterization and quantification of non-viral gene therapeutics, as well as their immunogenicity in vivo. However, the bioanalysis of non-viral based gene therapeutics continues to pose significant challenges due to their inherent complexity. Furthermore, potential interferences from sample matrices, anti-drug antibody (ADA), tissue sample variability, and co-administered medications further complicate the bioanalysis processes. In order to fulfill complex needs, different bioanalysis platforms can be applied, each of them has its own advantages/disadvantages and suitable application scenarios. Many cutting-edge or integrated bioanalysis methodologies are applied for the related bioanalysis for PK, biodistribution, efficacy and safety evaluations. The application of new platforms will also bring more regulatory challenges. Further detailed guidance will help to ensure the accuracy and robustness of the bioanalysis assay.

And to be noticed, the large-scale omics level tools including DNA-seq/RNA-seq, proteomics, and metabolomic could greatly accelerate early-stage research and development (R&D) for drug target identification and biomarker discovery to ensure the effectiveness and safe of the non-viral based gene therapeutics. For example, the microarray data from public repositories for 25 oligo drugs demonstrated about 25–130 genes undergo more than 50% changes for certain oligo treatment, revealing the drug-induced profile alternatives should be further evaluated by transcriptomics [181]. Beside the DNA/RNA sequencing technologies, the deep dive into the proteomics can provide further protein expression information to identify off-target effect associated with therapeutic modalities that induce gene silencing [182]. Besides the advance of mass spectrometer with better sensitivity and scan speed, the emerge of SomaLogic [183], Olink [184] and NULISA [185] large scale immune-linkage based assay platform further expend the application of proteomics. Moreover, the GenPro software developed for personalized protein databases (PPDs) may contribute to discriminate the single amino acid variants (SAAVs) that associate to the disease [186]. The single-molecule protein sequencing using nanopore-based strategy which can simultaneous discrimination of all 20 proteinogenic amino acids and their PTMs [187]. These technologies could provide insight for differentiating between modified protein isoforms, which could benefit the disease mechanism elucidation and biomarker discovery. In addition, machine learning and artificial intelligent (AI) approaches are also applied for the optimization of mRNA vaccine/therapeutics’ sequence for its better expression, stability and less immunogenicity [188]. The extensive application of AI and automation allows Moderna to ready its mRNA vaccine (mRNA-1273) from sequence selection to Phase 1 study dosing in 63 days. In summary, due to the nature of complexity of non-viral based gene therapeutics, more and more advanced and hybrid analytical platforms will be applied in the future. The next generation of AI algorisms and automation applications will further accelerate the non-viral based gene therapeutics development process.

5. Conclusion

In recent decades, research in gene therapeutics has advanced considerably, leading to enhanced insights into gene manipulation principles, the creation of novel delivery systems characterized by high efficiency and low toxicity, and the improvement of gene cargos potency and stability. Additionally, improved knowledge of the human genome and the discovery of new disease-related genes have provided more potential therapeutic targets for gene therapeutics. This has opened up possibilities for treating diseases that were previously considered undruggable using traditional approaches. In comparison to small molecule inhibitors and monoclonal antibodies, gene-editing drugs have the potential to permanently correct the disease genes. However, a key engineering challenge in gene therapy remains the establishment of safe and effective delivery pathway. Non-viral vectors are gaining traction as safer and more adaptable alternatives to viral vectors for administering gene therapies. They offer advantages such as a low risk of cytotoxicity, immunogenicity, carcinogenesis, and a flexible gene cargo packaging capacity.

Bioanalysis serves a vital function in the development of non-viral-based gene therapeutics. The composition of non-viral gene therapeutics varies, which could significantly impact the bioanalysis assay design. Generally, the bioanalysis strategies of the non-viral based gene therapeutics could be divided into four parts, the bioanalysis of the gene cargo, the bioanalysis of the vector carrier or its representative component, the bioanalysis of the target or expressed protein, and the immunogenicity evaluation. In each part, selecting the appropriate bioanalysis platform/method is necessary based on the property, size, and research stage of the test article. Each bioanalysis platform has its advantages and disadvantages. To ensure precise and effective PK/PD evaluation and immunogenicity assessment, it is essential to develop and implement a comprehensive bioanalytical strategy before initiating preclinical studies and clinical trials. With ongoing technological advancements, an array of bioanalysis strategies can be applied with better accuracy and sensitivity. Various platforms and techniques can be used, but the lack of detailed regulatory guidance on method validation in gene therapy may affect the quantification. In addition, familiarity with certain platforms may bias the selection process, even if other platforms may be more suitable for the analysis [171]. Despite the challenges, the success of two LNP-mRNA vaccines, BNT162b2 and mRNA-1273 has sparked optimism for the discovery of more innovative and safe non-viral gene therapeutics in the future. The increasing trend of gene therapeutics will open more opportunities for bioanalytical scientists, technology providers, and regulatory agencies to work together to establish the best practices aimed at navigating technical and regulatory challenges. This collaborative effort will be crucial in ensuring the continued advancement and success of gene therapeutic innovations.

Funding Statement

This paper was not funded.

Article highlights

• Non-viral vectors are gaining traction as safer and more adaptable alternatives to viral vectors for administering gene therapies. The bioanalysis of non-viral gene therapeutics is poised to offer technical support and a research foundation for gene therapeutic innovations.

• Various bioanalytical techniques are summarized for assessing pharmacokinetics (PK), biodistribution, and immunogenicity of non-viral gene therapeutics, with key focus areas including the analysis of gene cargo, vector, expressed protein, and the evaluation of potential immune responses.

• This work presents perspectives on several related areas, including the selection and evaluation of biomarkers, the challenges and solutions encountered in biological sample preparation, and the current regulatory perspective. By discussing these critical issues, it contributes to the advancement of research methodologies in the field.

Disclaimer

The information in this article is intended for informational purposes only and is not a substitute for professional medical advice. Please note that WuXi AppTec DMPK does not offer gene therapy or gene sequencing services.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.