Abstract
Cellular delivery of DNA vectors for the expression of therapeutic proteins is a promising approach to treat monogenic disorders or cancer. Significant efforts in a preclinical and clinical setting have been made to develop potent nonviral gene delivery systems based on lipoplexes composed of permanently cationic lipids. However, transfection efficiency and tolerability of such systems are in most cases not satisfactory. Here, we present a one-pot combinatorial method based on double-reductive amination for the synthesis of short-chain aminolipids. These lipids can be used to maximize the DNA vector delivery when combined with the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). We incorporated various aminolipids into such lipoplexes to complex minicircle DNA and screened these systems in a human liver-derived cell line (HuH7) for gene expression and cytotoxicity. The lead aminolipid AL-A12 showed twofold enhanced gene delivery and reduced toxicity compared to the native DOTAP:cholesterol lipoplexes. Moreover, AL-A12-containing lipoplexes enabled enhanced transgene expression in vivo in the zebrafish embryo model.
Highlights
Delivery of genetic material to diseased cells is of growing clinical interest
Lipoplexes allow for a straightforward transfer “from bench to large-scale production” compared to viral vectors[18] without the risk for insertion of genetic material into the host’s genome as shown for adeno-associated virus vectors.[19−21] cationic lipids overcome some of the limitations described for viral vectors, they encompass some disadvantages: The permanent positive charge of cationic lipids is associated with cytotoxicity,[22] and lipoplex systems generally lack efficiency compared to viral vectors.[23,24]
In addition to aliphatic amines, heterocyclic amines were selected as headgroups based on previous reports that inclusion of aromatic or heterocyclic rings into transfection reagents can increase the transfection efficiency.[41−45] To test whether these principles apply to DNA vector delivery based on dioleoyl-3-trimethylammonium propane and cholesterol (DOTAP):chol lipoplexes, different building blocks were selected and combined to afford the corresponding aminolipids
Summary
Delivery of genetic material to diseased cells is of growing clinical interest. Many inherited liver diseases including Crigler−Najjar syndrome or Gilbert’s syndrome are associated with genetic defects leading to loss of function of proteins or low levels of enzymatic activity.[1−8] Gene therapy enables the introduction of genetic material (e.g., DNA vectors) encoding for a therapeutic protein, thereby restoring physiological functions. In 2017, lipofection (i.e., the introduction of exogenous genetic material by means of lipid-based transfection reagents) accounted for only 4.5% of all clinical trials involving gene therapy.[25] As one of the exceptions, 1,2-dioleoyl-3-trimethylammonium propane and cholesterol (DOTAP:chol)-based lipoplexes have been clinically investigated for the treatment of various diseases including lung cancer, breast cancer, prostate cancer, or hepatocellular carcinoma.[9,13,15,16,26,27] The cationic charge of DOTAP:chol lipoplexes enables facilitated cellular interactions and accounts for decreased cell viability.[24,28] as observed for many lipoplexes, the use of DOTAP:chol systems is limited by poor transfection efficiency and cytotoxic effects.[29−32]
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