Hereditary cutaneous disorders contain a diverse array of diseases stemming from genetic aberrations. Two principal categories encompass a substantial patient population in clinical practice, yet their improvement remains challenging through conventional treatment strategies. Recessive dystrophic epidermolysis bullosa (RDEB) (OMIM#226600) is a hereditary disorder categorized under epidermolysis bullosa (EB), distinguished by manifestations such as skin fragility, blister formation, milia, and scarring [1]. The etiology of RDEB is rooted in mutations within the COL7A1 gene, responsible for encoding type VII collagen (C7) [2]. C7 assumes a pivotal role as a major constituent of anchoring fibrils (AFs), crucial structures facilitating epidermal adhesion to the dermis at the dermal-epidermal junction [3]. Another genodermatosis of significance is ichthyosis, with lamellar ichthyosis (LI) (OMIM#242300) representing a subtype within the broader category of autosomal recessive congenital ichthyosis (ARCI) [4,5]. Germline mutations in the TGM1 gene predominate affects over 15% of ARCI cases [6]. Transglutaminase-1 protein, encoded by the TGM1 gene, plays a critical role in the formation of the cornified cell envelope, an indispensable component for maintaining the integrity of the skin barrier [7,8].

Regrettably, prevailing therapeutic modalities for addressing hereditary cutaneous disorders offer solely symptomatic relief [[9], [10], [11]]. Individuals with severe skin involvement find conventional medications inadequate in restoring epidermal barrier function, and such treatments pose the risk of significant complications [12]. Consequently, there exists a substantial unmet need for a therapeutic intervention capable of molecularly correcting the underlying genetic deficiency.

Gene therapy involves the introduction of functional genes into specific cells or tissues to manipulate or modulate gene expression, representing one of the most promising treatment approaches for a range of inherited disorders [[13], [14], [15]]. Accessibility for gene delivery, clinical evaluation, and topical modulation of gene expression render the skin a very attractive tissue for therapeutic gene delivery. However, there are several key hurdles to be overcome before cutaneous gene therapy becomes a viable clinical option [16]. The widespread clinical application of gene therapy is impeded by the scarcity of secure and efficient gene delivery vehicles. Non-viral vectors, with a focus on cationic polymers and liposomes, have exhibited robust gene loading capacity, safety, practicality, and simplicity in preparation [17,18].

In 2000, Langer et al. innovatively synthesized linear poly(β-amino ester) (LPAE), identifying it as a highly effective non-viral gene vector using the “A2 + B2” Michael addition reaction with diamine and diacrylate monomers [[19], [20], [21]]. In contrast to linear polymers, highly branched polymers possess a three-dimensional structure and multiple functionalized sites, significantly enhanced the interaction between polymer vectors and DNA [[22], [23], [24]]. Afterwards, Zhou et al. utilized a one-pot “A2 + B3 + C2” Michael addition strategy to synthesize a series of highly branched poly(β-amino ester)s (HPAEs) for efficient DNA delivery both in vitro and in vivo [25]. The optimized HPAEs demonstrated a remarkable up to thousands-fold in gene transfection efficiency compared to their LPAEs in vitro [26,27]. Notably, HPAEs demonstrated proficient delivery of COL7A1 plasmids, leading to the reinstatement of high-level C7 protein expression in RDEB mouse. Furthermore, the top-performing HPAEs efficiently mediate the transfection of nerve growth factor-encoding plasmids, fostering neurite outgrowth and nerve cell recovery in nerve-related cells, ultimately inducing the growth of nerve protrusion [28,29].

Nonetheless, employing the widely applicable one-pot Michael addition strategy, all HPAEs exhibited significant molecular weight distribution (MWD), as indicated by elevated polydispersity index (Đ) values reaching up to 12.0 [[30], [31], [32]]. Throughout the gene transfection process, there are multiple extracellular and intracellular barriers during the gene transfection process, one can envisage that different molecular weight (MW) would lead HPAEs to exhibit distinct capability of navigating these barriers and ultimately mediating diverse level of transfection activity [33]. A portion of the components with MW out of a moderate range would be low in gene transfection and merely contribute to increasing the cytotoxicity of HPAEs, which was unfavorable to the clinical approval of HPAE in gene therapy for inherited diseases [[33], [34]].

Therefore, the goal of this study is to establish a facile elution fractionation strategy for isolating HPAE components with a narrow MWD, initially evaluate the expression efficiency of optimized polymer-mediated TGM1 and COL7A1 plasmids, along with exploring the therapeutic potential for RDEB and ARCI. To achieve this, we initially selected bisphenol A polyethylene glycol diether diacrylate (BEDA), trimethylolpropane triacrylate (TMPTA), 4-amino-1-butanol (S4), and N-(3-Aminopropyl)morpholine (MPA) as end-capping monomers to synthesize HAPEs with Mw of 23.7 kg/mol, and a wide Đ of 3.8 (Fig. 1a). Subsequently, a step-by-step elution fractionation strategy was employed to fractionate HPAEs, isolating components with different MW but a narrow Đ (Fig. 1b). Through meticulous adjustments in the precipitant's polarity and the solubility of distinct components, a sequential isolation of a series of HPAEs was achieved, with MW ranging from 4.4 kg/mol to 131.8 kg/mol and a dispersity index (Đ) of <2.0, at different volume-to- volume (v/v) ratios of acetone/diethyl ether solvent mixture. Gene transfection results revealed that the intermediate MW HPAEs, HPAE20.6k, exhibited the highest gene transfection efficiency (46.4%) and a strongest mean fluorescence intensity (143,032 RLU), compared to other isolated components and the crude product, which was 12.5-fold and 5.5-fold higher than that mediated by low MW HPAE4.4k, at the lowest w/w ratio of 20:1. Furthermore, the physicochemical analysis of isolated components and formulated polyplexes indicated that the intermediate MW and narrow MWD endowed HPAE20.6k with enhanced DNA binding affinity, resulting in the formulation of polyplexes with smaller size and a suitable positive zeta potential. Importantly, we demonstrated that best-performing HPAE20.6k, with its intermediate MW and narrow MWD, effectively delivered COL7A1 (15,974 bp) and TGM1 (7181 bp) plasmids, promoting the efficient expression of C7 and transglutaminase-1 proteins in skin cells, which highlighted their potential application in the treatment of RDEB and ARCI. Our study establishes a straightforward step-by-step elution fractionation strategy for the development of HPAE gene delivery vectors, expediting their clinical translation in inherited skin diseases.