Biodegradable Lipid Nanoparticles Research Articles

Unleashing the Power of Proenzyme Delivery for Targeted Therapeutic Applications Using Biodegradable Lipid Nanoparticles.

Proenzymes, functioning as inactive precursor forms of enzymes, hold significant promise for regulating essential biological processes. Their inherent property of latency, remaining inert until they arrive at the intended site of action, positions them as particularly promising candidates for the development of targeted therapeutics. Despite this potential, the therapeutic potential of proenzymes is challenged by designing proenzymes with excellent selectivity for disease cells. This limitation is further exacerbated by the inability of proenzymes to spontaneously cross the cell membrane, a biological barrier that impedes the cellular internalization of exogenous macromolecules. Therefore, efficacious intracellular delivery is paramount to unlocking the full therapeutic potency of proenzymes.In this Account, we first elucidate our recent advancements made in designing biodegradable lipid nanoparticles (LNPs) for the cell-specific delivery of biomacromolecules, including proteins and nucleic acids. Using a strategy of parallel synthesis, we have constructed an extensive library of ionizable lipids, each integrated with different biodegradable moieties. This combinatorial approach has led to the identification of LNPs that are particularly efficacious for the delivery of biomacromolecules specifically to tumor cells. This innovation capitalizes on the unique intracellular environment of cancer cells to control the degradation of LNPs, thereby ensuring the targeted release of therapeutics within tumor cells. Additionally, we discuss the structure-activity relationship governing the delivery efficacy of these LNPs and their applicability in regulating tumor cell signaling, specifically through the delivery of bacterial effector proteins.In the second segment, we aim to provide an overview of our recent contributions to the field of proenzyme design, where we have chemically tailored proteins to render them responsive to the unique milieu of tumor cells. Specifically, we elaborate on the chemical principles employed to modify proteins and DNAzymes, thereby priming them for activation in the presence of NAD(P)H:quinone oxidoreductase 1 (NQO1), an enzyme that is prevalently upregulated within tumor cells. We summarize the methodologies for intracellular delivery of these proenzymes using biodegradable LNPs, both in vitro and in vivo. The concomitant intracellular delivery and activation of proenzymes are examined in the context of enhanced therapeutic outcomes and targeted CRISPR/Cas9 genome editing.In conclusion, we offer a perspective on the chemical principles that could be leveraged to optimize LNPs for tissue-specific delivery of proenzymes. We also explore chemical strategies for the irreversible modulation of proenzyme activity within living cells and in vivo. Through this discussion, we provide insights into potential avenues for overcoming existing limitations and enhancing the delivery of proenzymes using LNPs, particularly for developing tumor-targeted therapies and genome editing applications.

Intracellular delivery of bacterial effectors for cancer therapy using biodegradable lipid nanoparticles.

Bacterial effector proteins are virulence factors that are secreted and mediate orthogonal post-translational modifications of proteins that are not found naturally in mammalian systems. They hold great promise for developing biotherapeutics by regulating malignant cell signaling in a specific and targeted manner. However, delivering bacterial effectors into disease cells poses a significant challenge to their therapeutic potential. In this study, we report on the design of a combinatorial library of bioreducible lipid nanoparticles containing disulfide bonds for highly efficient bacterial effector delivery and potential cancer therapy. A leading lipid, PPPDA-O16B, identified from the library, can encapsulate and deliver DNA plasmids into cells. The gene cargo is released in response to the reductive cellular environment that is upregulated in cancer cells, leading to enhanced gene delivery and protein expression efficiency. Furthermore, we demonstrate that PPPDA-O16B can deliver the bacterial effector protein, DUF5, to degrade mutant RAS and inactivate downstream MAPK signaling cascades to suppress cancer cell growth in vitro and in tumor-bearing mouse xenografts. This strategy of delivering bacterial effectors using biodegradable lipid nanoparticles can be expanded for cancer cell signaling regulation and antitumor studies.

Development of amino acid-modified biodegradable lipid nanoparticles for siRNA delivery

The use of siRNA therapeutics to treat cancer is a very promising approach. However, specific delivery of siRNAs to tumors remains a major challenge. The recent success of siRNA delivery to the liver has incentivized the development of biomaterials for siRNA delivery into tumors. Here, we report a new class of amino acid-modified lipids for siRNA delivery to cancer cells. Eight lipids were developed by headgroup modification with histidine and lysine. The lipids were screened in PC3-luciferase stable cells for gene silencing and cellular cytotoxicity study. The best lipid LHHK shows a pKa of 6.08, which is within the optimal pKa range of lipid nanoparticles (LNPs) for siRNA delivery. The LHHK LNP protects siRNA from serum degradation for up to 24 h and shows higher endosomal release and better cellular uptake compared to other lysine-modified lipids in PC3 cells. The LHHK LNP exhibits significant silencing activity of IKKα and IKBKE in prostate cancer and pancreatic cancer, respectively. Moreover, the LHHK LNP encapsulating IKBKE siRNA inhibits cell proliferation of pancreatic cancer cells and suppresses the tumor progression in a pancreatic cancer mouse model. Statement of significanceLipid nanoparticle (LNP) is a promising platform for siRNA delivery. However, LNP is generally associated with high systemic toxicity. As a result, efficient and biodegradable lipids are highly needed for siRNA-based cancer therapy. Herein, we develop amino acid-modified biodegradable lipids. These lipids show very low cellular toxicity and high transfection efficiency. The best lipid LHHK shows a pKa of 6.08, which is within the optimal pKa range of LNPs for siRNA delivery. The LHHK LNP efficiently silences IKKα and IKBKE in prostate and pancreatic cancer, respectively. Moreover, the LHHK LNP encapsulating IKBKE siRNA inhibits cell proliferation and suppresses tumor growth of pancreatic cancer in vivo. These results suggest that amino acid-modified lipids possess a great potential for siRNA delivery in cancer therapy.

Intracellular Delivery of Glutathione Peroxidase Degrader Induces Ferroptosis In Vivo.

Ferroptosis is a new form of regulated, non-apoptotic cell death driven by iron-dependent phospholipid peroxidation. Its therapeutic potential is however, greatly limited by the low efficiency of regulating cell ferroptosis in vivo. Herein, we report a PROTAC-based protein degrader that depletes endogenous glutathione peroxidase 4 (GPX4) and induces cancer cell ferroptosis. We demonstrate that a rationally designed GPX4 degrader, dGPX4, can deplete tumor cell GPX4 via proteasomal protein degradation, showing a five-fold enhancement of ferroptosis induction efficiency compared to that of GPX4 inhibition using ML162. Moreover, we show that the intracellular delivery of dGPX4 using biodegradable lipid nanoparticles (dGPX4@401-TK-12) induces cell-selective ferroptosis by targeting cancer cell microenvironment. The in vivo administration of dGPX4@401-TK-12 effectively suppresses tumor growth without appreciable side effects. We anticipate the protein degradation strategy described herein could be easily expanded to other essential regulatory proteins of ferroptosis for developing targeted cancer therapeutics.

Intracellular Delivery of Glutathione Peroxidase Degrader Induces Ferroptosis In Vivo

AbstractFerroptosis is a new form of regulated, non‐apoptotic cell death driven by iron‐dependent phospholipid peroxidation. Its therapeutic potential is however, greatly limited by the low efficiency of regulating cell ferroptosis in vivo. Herein, we report a PROTAC‐based protein degrader that depletes endogenous glutathione peroxidase 4 (GPX4) and induces cancer cell ferroptosis. We demonstrate that a rationally designed GPX4 degrader, dGPX4, can deplete tumor cell GPX4 via proteasomal protein degradation, showing a five‐fold enhancement of ferroptosis induction efficiency compared to that of GPX4 inhibition using ML162. Moreover, we show that the intracellular delivery of dGPX4 using biodegradable lipid nanoparticles (dGPX4@401‐TK‐12) induces cell‐selective ferroptosis by targeting cancer cell microenvironment. The in vivo administration of dGPX4@401‐TK‐12 effectively suppresses tumor growth without appreciable side effects. We anticipate the protein degradation strategy described herein could be easily expanded to other essential regulatory proteins of ferroptosis for developing targeted cancer therapeutics.

A Combinatorial Library of Biodegradable Lipid Nanoparticles Preferentially Deliver mRNA into Tumor Cells to Block Mutant RAS Signaling

AbstractMessenger RNA (mRNA) is an emerging class of biotherapeutics for vaccine development and genome editing. Efficacious delivery and control of mRNA functionality selectively to disease cells remains the major challenge in developing mRNA therapeutics. Herein, reactive oxygen species (ROS)‐degradable lipid nanoparticles containing a thioketal (TK) moiety to deliver mRNA into cells are reported, selectively releasing mRNA in tumor cells for enhanced gene expression. By screening a library of parallelly synthesized ROS‐degradable lipids, it has been identified that BAmP‐TK‐12 delivers mRNA one‐fold more potent in tumor cells than in non‐cancerous cells. Furthermore, the delivery of mRNA encoding DUF5, a bacterial‐derived RAS protease using BAmP‐TK‐12 enables generic depletion of mutant RAS of tumor cells, showing a significantly improved antitumor effect than small molecule‐based RAS inhibitor. It has been believed that the strategy of tumor cell‐selective mRNA delivery using ROS‐degradable lipid nanoparticles can be expanded to the broad range of bacterial effectors for rewiring cancer cell signaling and developing advanced biotherapeutics.

Lipid Nanoparticles as a Promising Drug Delivery Carrier for Topical Ocular Therapy-An Overview on Recent Advances.

Due to complicated anatomical and physical properties, targeted drug delivery to ocular tissues continues to be a key challenge for formulation scientists. Various attempts are currently being made to improve the in vivo performance of therapeutic molecules by encapsulating them in various nanocarrier systems or devices and administering them via invasive/non-invasive or minimally invasive drug administration methods. Biocompatible and biodegradable lipid nanoparticles have emerged as a potential alternative to conventional ocular drug delivery systems to overcome various ocular barriers. Lipid-based nanocarrier systems led to major technological advancements and therapeutic advantages during the last few decades of ocular therapy, such as high precorneal residence time, sustained drug release profile, minimum dosing frequency, decreased drug toxicity, targeted site delivery, and, therefore, an improvement in ocular bioavailability. In addition, such formulations can be given as fine dispersion in patient-friendly droppable preparation without causing blurred vision and ocular sensitivity reactions. The unique advantages of lipid nanoparticles, namely, solid lipid nanoparticles, nanostructured lipid carriers, nanoemulsions, and liposomes in intraocular targeted administration of various therapeutic drugs are extensively discussed. Ongoing and completed clinical trials of various liposome-based formulations and various characterization techniques designed for nanoemulsion in ocular delivery are tabulated. This review also describes diverse solid lipid nanoparticle preparation methods, procedures, advantages, and limitations. Functionalization approaches to overcome the drawbacks of lipid nanoparticles, as well as the exploration of new functional additives with the potential to improve the penetration of macromolecular pharmaceuticals, would quickly progress the challenging field of ocular drug delivery systems.

Developing Biodegradable Lipid Nanoparticles for Intracellular mRNA Delivery and Genome Editing.

Since the U.S. Food and Drug Administration (FDA) granted emergency use authorization for two mRNA vaccines against SARS-CoV-2, mRNA-based technology has attracted broad attention from the scientific community to investors. When delivered intracellularly, mRNA has the ability to produce various therapeutic proteins, enabling the treatment of a variety of illnesses, including but not limited to infectious diseases, cancers, and genetic diseases. Accordingly, mRNA holds significant therapeutic potential and provides a promising means to target historically hard-to-treat diseases. Current clinical efforts harnessing mRNA-based technology are focused on vaccination, cancer immunotherapy, protein replacement therapy, and genome editing. The clinical translation of mRNA-based technology has been made possible by leveraging nanoparticle delivery methods. However, the application of mRNA for therapeutic purposes is still challenged by the need for specific, efficient, and safe delivery systems.This Account highlights key advances in designing and developing combinatorial synthetic lipid nanoparticles (LNPs) with distinct chemical structures and properties for in vitro and in vivo intracellular mRNA delivery. LNPs represent the most advanced nonviral nanoparticle delivery systems that have been extensively investigated for nucleic acid delivery. The aforementioned COVID-19 mRNA vaccines and one LNP-based small interfering RNA (siRNA) drug (ONPATTRO) have received clinical approval from the FDA, highlighting the success of synthetic ionizable lipids for in vivo nucleic acid delivery. In this Account, we first summarize the research efforts from our group on the development of bioreducible and biodegradable LNPs by leveraging the combinatorial chemistry strategy, such as the Michael addition reaction, which allows us to easily generate a large set of lipidoids with diverse chemical structures. Next, we discuss the utilization of a library screening strategy to identify optimal LNPs for targeted mRNA delivery and showcase the applications of the optimized LNPs in cell engineering and genome editing. Finally, we outline key challenges to the clinical translation of mRNA-based therapies and propose an outlook for future directions of the chemical design and optimization of LNPs to improve the safety and specificity of mRNA drugs. We hope this Account provides insight into the rational design of LNPs for facilitating the development of mRNA therapeutics, a transformative technology that promises to revolutionize future medicine.

Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model

Therapeutic targeting of injuries that require transient restoration of proteins by mRNA delivery is an attractive approach that, until recently, has remained poorly explored. In this study, we examined the therapeutic utility of mRNA delivery for liver fibrosis and cirrhosis. Specifically, we aimed to demonstrate the therapeutic efficacy of human hepatocyte nuclear factor alpha (HNF4A) mRNA in mouse models of fibrosis and cirrhosis. We investigated restoration of hepatocyte functions by HNF4A mRNA transfection invitro, and analyzed the attenuation of liver fibrosis and cirrhosis in multiple mouse models, by delivering hepatocyte-targeted biodegradable lipid nanoparticles (LNPs) encapsulating HNF4A mRNA. To identify potential mechanisms of action, we performed microarray-based gene expression profiling, single-cell RNA sequencing, and chromatin immunoprecipitation. We used primary liver cells and human liver buds for additional functional validation. Expression of HNF4A mRNA led to restoration of the metabolic activity of fibrotic primary murine and human hepatocytes invitro. Repeated invivo delivery of LNP-encapsulated HNF4A mRNA induced a robust inhibition of fibrogenesis in 4 independent mouse models of hepatotoxin- and cholestasis-induced liver fibrosis. Mechanistically, we discovered that paraoxonase 1 is a direct target of HNF4A and it contributes to HNF4A-mediated attenuation of liver fibrosis via modulation of liver macrophages and hepatic stellate cells. Collectively, our findings provide the first direct preclinical evidence of the applicability of HNF4A mRNA therapeutics for the treatment of fibrosis in the liver. Liver fibrosis and cirrhosis remain unmet medical needs and contribute to high mortality worldwide. Herein, we take advantage of a promising therapeutic approach to treat liver fibrosis and cirrhosis. We demonstrate that restoration of a key gene, HNF4A, via mRNA encapsulated in lipid nanoparticles decreased injury in multiple mouse models of fibrosis and cirrhosis. Our study provides proof-of-concept that mRNA therapy is a promising strategy for reversing liver fibrosis and cirrhosis.

Dual mRNA therapy restores metabolic function in long-term studies in mice with propionic acidemia

Propionic acidemia/aciduria (PA) is an ultra-rare, life-threatening, inherited metabolic disorder caused by deficiency of the mitochondrial enzyme, propionyl-CoA carboxylase (PCC) composed of six alpha (PCCA) and six beta (PCCB) subunits. We herein report an enzyme replacement approach to treat PA using a combination of two messenger RNAs (mRNAs) (dual mRNAs) encoding both human PCCA (hPCCA) and PCCB (hPCCB) encapsulated in biodegradable lipid nanoparticles (LNPs) to produce functional PCC enzyme in liver. In patient fibroblasts, dual mRNAs encoded proteins localize in mitochondria and produce higher PCC enzyme activity vs. single (PCCA or PCCB) mRNA alone. In a hypomorphic murine model of PA, dual mRNAs normalize ammonia similarly to carglumic acid, a drug approved in Europe for the treatment of hyperammonemia due to PA. Dual mRNAs additionally restore functional PCC enzyme in liver and thus reduce primary disease-associated toxins in a dose-dependent manner in long-term 3- and 6-month repeat-dose studies in PA mice. Dual mRNAs are well-tolerated in these studies with no adverse findings. These studies demonstrate the potential of mRNA technology to chronically administer multiple mRNAs to produce large complex enzymes, with applicability to other genetic disorders.

Efficient Delivery of Antisense Oligonucleotides Using Bioreducible Lipid Nanoparticles In Vitro and In Vivo

The efficient delivery of antisense oligonucleotides (ASOs) to the targeted cells and organs remains a challenge, in particular, in vivo. Here, we investigated the ability of a library of biodegradable lipid nanoparticles (LNPs) in delivering ASO to both cultured human cells and animal models. We first identified three top-performing lipids through in vitro screening using GFP-expressing HEK293 cells. Next, we explored these three candidates for delivering ASO to target proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA in mice. We found that lipid 306-O12B-3 showed efficiency with the median effective dose (ED50) as low as 0.034 mg·kg-1, which is a notable improvement over the efficiency reported in the literature. No liver or kidney toxicity was observed with a dose up to 5 mg·kg-1 of this ASO/LNP formulation. The biodegradable LNPs are efficient and safe in the delivery of ASO and pave the way for clinical translation.

Systemic Messenger RNA Therapy as a Treatment for Methylmalonic Acidemia

Isolated methylmalonic acidemia/aciduria (MMA) is a devastating metabolic disorder with poor outcomes despite current medical treatments. Like other mitochondrial enzymopathies, enzyme replacement therapy (ERT) is not available, and although promising, AAV gene therapy can be limited by pre-existing immunity and has been associated with genotoxicity in mice. To develop a new class of therapy for MMA, we generated a pseudoU-modified codon-optimized mRNA encoding human methylmalonyl-CoA mutase (hMUT), the enzyme most frequently mutated in MMA, and encapsulated it into biodegradable lipid nanoparticles (LNPs). Intravenous (i.v.) administration of hMUT mRNA in two different mouse models of MMA resulted in a 75%-85% reduction in plasma methylmalonic acid and was associated with increased hMUT protein expression and activity in liver. Repeat dosing of hMUT mRNA reduced circulating metabolites and dramatically improved survival and weight gain. Additionally, repeat i.v. dosing did not increase markers of liver toxicity or inflammation in heterozygote MMA mice.

Interactions of the antimicrobial peptide nisin Z with conventional antibiotics and the use of nanostructured lipid carriers to enhance antimicrobial activity

Antimicrobial resistance is an imminent threat to the effective prevention and treatment of bacterial infections and alternative antimicrobial strategies are desperately needed. Antimicrobial peptides (AMPs) may be promising alternatives to current antibiotics or act as adjuvants to enhance antibiotic potency. Additionally, the use of biodegradable lipid nanoparticles can enhance the antibacterial activity of antibiotics and antimicrobial peptides. In this study, the interaction of the AMPs, nisin Z and melittin, with conventional antibiotics was investigated on Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli. The effectiveness of nanostructured lipid carriers (NLCs) for the entrapment of nisin Z was also evaluated. Findings revealed that nisin Z exhibited additive interactions with numerous conventional antibiotics. Notable synergism was observed for novobiocin-nisin Z combinations. The addition of the non-antibiotic adjuvant EDTA significantly improved the antimicrobial activity of free nisin Z towards E.coli. NLCs containing nisin Z were effective against Gram-positive species at physiological pH, with an increase in effectiveness in the presence of EDTA. Results indicate that nisin Z may be advantageous as an adjuvant in antimicrobial chemotherapy, while contributing in the battle against antibiotic resistance. NLCs have the potential to enhance the antibacterial activity of nisin Z towards Gram-positive bacterial species associated with skin infections.

Nanoparticles encapsulated with resveratrol induce browning of white adipocytes

Functional brown adipose tissue, including classical brown adipocytes and brown‐like or beige adipocytes, which develop within white adipose tissue (WAT) in response to various stimuli, has emerged as novel targets for obesity treatment and prevention. Through browning process, beige adipocytes are induced to express UCP‐1 and become capable of thermogenesis. Identifying nutritional strategies that increase UCP‐1 and induce browning within white adipocytes is key to unlocking browning potential. Resveratrol (Res) is a well‐known polyphenol found in the skins of grapes and other fruits such as berries with anti‐inflammatory, antioxidant, and anti‐carcinogenic properties. Nanotechnology has shown great promise in encapsulating polyphenols, such as Res, to increase their solubility and stability, consequently, bioactivities. In this study, Res encapsulated in biocompatible and biodegradable lipid nanoparticles (Nano‐Res) or liposomes (Lipo‐Res), along with free Res (Res) and their respective controls (Nano‐Void or Lipo‐Void respectively) were tested at three doses (5, 10, 20 μM) in 3T3‐L1 mouse pre‐adipocytes in a 7‐day differentiation process. At the end of differentiation, the cells were subjected to thermogenic activation by isoproterenol (ISO), a β‐adrenergic receptor agonist, or the control for 6 hours. Cytotoxicity of the treatments and mRNA expression of UCP‐1, brown (PPARγ, PGC1α, PRDM16, and Cidea), beige (CD137 and Tmem26), and white marker genes (IGFBP3), as well as mitochondria related genes (Cox4a, Uqrch, Nrf, and Tfam) were measured. We report that none of the treatments showed signs of cytotoxicity at 24‐hrs and minimal toxicity at 72‐hrs. Lipo‐Res and Nano‐Res significantly induced dose‐dependent increase of UCP‐1 mRNA expression under ISO stimulated conditions compared to the controls (p<0.05). At the low dose of 5 μM Lipo‐Res significantly stimulated UCP‐1 to a higher level than free Res (p<0.05). Under both basal (non‐ISO stimulated) and ISO stimulated conditions, both Lipo‐Res and Nano‐Res dose‐dependently decreased WAT marker IGFBP3 mRNA (p<0.05) while simultaneously increasing mRNA of beige marker CD137 and brown markers PRDM16 (p<0.05). Interestingly, Lipo‐Res significantly increased Cidea expression in ISO‐stimulated conditions (p<0.05); however, Nano‐Res (and Nano‐Void) decreased Cidea expression (p<0.05). Other beige markers and mitochondrial structural genes were increased by encapsulated Res, but did not reach significance compared to the controls. Taken together, our results demonstrate that nanoencapsulation enhances Res's bioactivities in inducing browning of white adipocytes. Our results point towards promising nutritional strategies for obesity treatment and prevention.Support or Funding InformationThe work has been supported by NIH 1R15AT008733 award.

Biodegradable lipid nanoparticles induce a prolonged RNA interference-mediated protein knockdown and show rapid hepatic clearance in mice and nonhuman primates

Lipid nanoparticles based on ionizable lipids have been clinically validated as a means of delivery for RNA interference (RNAi) therapeutics. The ideal properties of RNAi carriers are efficient delivery of oligonucleotides into target cells and rapid elimination after the function is performed. Here, we report that degradable lipid nanoparticles are effective carriers of small interfering RNA (siRNA) and have a high therapeutic index. The newly developed degradable lipid nanoparticles carrying siRNA showed potent gene-silencing activity in mouse hepatocytes (ED50≈0.02mg/kg siRNA). The ester bond in the lipid tail was hydrolyzed in the liver, resulting in rapid metabolism of the lipid. Toxicity assays showed that the degradable lipid was well-tolerated at siRNA doses of up to 16mg/kg in rats (over 800-fold higher than ED50). A single intravenous injection of siRNA targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) in cynomolgus monkeys resulted in more than 90% protein silencing, and a 50% decrease in plasma low-density lipoprotein (LDL) cholesterol, with a measurable reduction for 2 months. Moreover, quantification of lipids in liver biopsies revealed rapid hepatic clearance of the degradable lipid in nonhuman primates. These degradable lipid nanoparticles with a high therapeutic index hold promise for RNA-based treatments.

The effect of dissolved volatiles on magmatic heat sources at intrusive contacts

Decades of intense research in molecular biology and biochemistry are fructifying in the emergence of therapeutic messenger RNAs (mRNA) as a new class of drugs. Synthetic mRNAs can be sequence optimised to improve translatability into proteins, as well as chemically modified to reduce immunogenicity and increase chemical stability using naturally occurring uridine modifications. These structural improvements, together with the development of safe and efficient vehicles that preserve mRNA integrity in circulation and allow targeted intracellular delivery, have paved the way for mRNA-based therapeutics. Indeed, mRNAs formulated into biodegradable lipid nanoparticles are currently being tested in preclinical and clinical studies for multiple diseases including cancer immunotherapy and vaccination for infectious diseases. An emerging application of mRNAs is the supplementation of proteins that are not expressed or are not functional in a regulated and tissue-specific manner. This so-called ‘protein replacement therapy’ could represent a solution for genetic metabolic diseases currently lacking effective treatments. Here we summarise this new class of drugs and discuss the preclinical evidence supporting the potential of liver-mediated mRNA therapy for three rare genetic conditions: methylmalonic acidaemia, acute intermittent porphyria and ornithine transcarbamylase deficiency.