Systemic delivery of mRNA and DNA to the lung using polymer-lipid nanoparticles.

Non-viral vectors offer the potential to deliver nucleic acids including mRNA and DNA into cells in vivo. However, designing materials that effectively deliver to target organs and then to desired compartments within the cell remains a challenge. Here we develop polymeric materials that can be optimized for either DNA transcription in the nucleus or mRNA translation in the cytosol. We synthesized poly(beta amino ester) terpolymers (PBAEs) with modular changes to monomer chemistry to investigate influence on nucleic acid delivery. We identified two PBAEs with a single monomer change as being effective for either DNA (D-90-C12-103) or mRNA (DD-90-C12-103) delivery to lung endothelium following intravenous injection in mice. Physical properties such as particle size or charge did not account for the difference in transfection efficacy. However, endosome co-localization studies revealed that D-90-C12-103 nanoparticles resided in late endosomes to a greater extent than DD-90-C12-103. We compared luciferase expression in vivo and observed that, even with nucleic acid optimized vectors, peak luminescence using mRNA was two orders of magnitude greater than pDNA in the lungs of mice following systemic delivery. This study indicates that different nucleic acids require tailored delivery vectors, and further support the potential of PBAEs as intracellular delivery materials.

mRNA Delivery for Therapeutic Anti-HER2 Antibody Expression In Vivo.

Antibody-based drugs are a leading class of biologics used to treat a variety of diseases, including cancer. However, wide antibody implementation is hindered by manufacturing challenges and high production cost. Use of in-vitro-transcribed mRNA (IVT-mRNA) for endogenous protein expression has the potential to circumvent many of the shortcomings of antibody production and therapeutic application. Here, we describe the development of an IVT-mRNA system for in vivo delivery of a humanized anti-HER2 (also known as ERBB2) antibody, trastuzumab, and demonstrate its anticancer activity. We engineered the IVT-mRNA sequence to maximize expression, then formulated the IVT-mRNA into lipid-based nanoparticles (LNPs) to protect the mRNA from degradation and enable efficient in vivo delivery. Systemic delivery of the optimized IVT-mRNA loaded into LNPs resulted in antibody serum concentrations of 45 ± 8.6 µg/mL for 14 days after LNP injection. Further studies demonstrated an improved pharmacokinetic profile of the produced protein compared to injection of trastuzumab protein. Finally, treatment of tumor-bearing mice with trastuzumab IVT-mRNA LNPs selectively reduced the volume of HER2-positive tumors and improved animal survival. Taken together, the results of our study demonstrate that using IVT-mRNA LNPs to express full-size therapeutic antibodies in the liver can provide an effective strategy for cancer treatment and offers an alternative to protein administration.

Improved Efficacy in a Fabry Disease Model Using a Systemic mRNA Liver Depot System as Compared to Enzyme Replacement Therapy.

Fabry disease is a lysosomal storage disorder caused by the deficiency of α-galactosidase A. Enzyme deficiency results in a progressive decline in renal and cardiac function, leading to cardiomyopathy and end-stage renal disease. Current treatments available, including enzyme replacement therapies, have provided significant benefit to patients; however, unmet medical needs remain. mRNA therapy, with drug-like properties, has the unique ability to produce therapeutic proteins endogenously. Here we describe the sustained delivery of therapeutic human α-galactosidase protein in vivo via nanoparticle-formulated mRNA in mouse and non-human primate, with a demonstration of efficacy through clinically relevant biomarker reduction in a mouse Fabry disease model. Multi-component nanoparticles formulated with lipids and lipid-like materials were developed for the delivery of mRNA encoding human α-galactosidase protein. Upon delivery of human GLA mRNA to mice, serum GLA protein levels reached as high as ∼1,330-fold over normal physiological values.

Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium.

Noninvasive aerosol inhalation is an established method of drug delivery to the lung, and remains a desirable route for nucleic-acid-based therapeutics. In vitro transcribed (IVT) mRNA has broad therapeutic applicability as it permits temporal and dose-dependent control of encoded protein expression. Inhaled delivery of IVT-mRNA has not yet been demonstrated and requires development of safe and effective materials. To meet this need, hyperbranched poly(beta amino esters) (hPBAEs) are synthesized to enable nanoformulation of stable and concentrated polyplexes suitable for inhalation. This strategy achieves uniform distribution of luciferase mRNA throughout all five lobes of the lung and produces 101.2 ng g-1 of luciferase protein 24 h after inhalation of hPBAE polyplexes. Importantly, delivery is localized to the lung, and no luminescence is observed in other tissues. Furthermore, using an Ai14 reporter mouse model it is identified that 24.6% of the total lung epithelial cell population is transfected after a single dose. Repeat dosing of inhaled hPBAE-mRNA generates consistent protein production in the lung, without local or systemic toxicity. The results indicate that nebulized delivery of IVT-mRNA facilitated by hPBAE vectors may provide a clinically relevant delivery system to lung epithelium.

Optimization of a Degradable Polymer-Lipid Nanoparticle for Potent Systemic Delivery of mRNA to the Lung Endothelium and Immune Cells.

mRNA therapeutics hold great potential for treating a variety of diseases through protein-replacement, immunomodulation, and gene editing. However, much like siRNA therapy the majority of progress in mRNA delivery has been confined to the liver. Previously, we demonstrated that poly(ß-amino esters), a class of degradable polymers, are capable of systemic mRNA delivery to the lungs in mice when formulated into nanoparticles with poly(ethylene glycol)-lipid conjugates. Using experimental design, a statistical approach to optimization that reduces experimental burden, we demonstrate herein that these degradable polymer-lipid nanoparticles can be optimized in terms of polymer synthesis and nanoparticle formulation to achieve a multiple order-of-magnitude increase in potency. Furthermore, using genetically engineered Cre reporter mice, we demonstrate that mRNA is functionally delivered to both the lung endothelium and pulmonary immune cells, expanding the potential utility of these nanoparticles.

Customizable Lipid Nanoparticle Materials for the Delivery of siRNAs and mRNAs.

RNAs are a promising class of therapeutics given their ability to regulate protein concentrations at the cellular level. Developing safe and effective strategies to deliver RNAs remains important for realizing their full clinical potential. Here, we develop lipid nanoparticle formulations that can deliver short interfering RNAs (for gene silencing) or messenger RNAs (for gene upregulation). Specifically, we study how the tail length, tail geometry, and linker spacing in diketopiperazine lipid materials influences LNP potency with siRNAs and mRNAs. Eight lipid materials are synthesized, and 16 total formulations are screened for activity in vitro; the lead material is evaluated with mRNA for in vivo use and demonstrates luciferase protein expression in the spleen. In undertaking this approach, not only do we develop synthetic routes to delivery materials, but we also reveal structural criteria that could be useful for developing next-generation delivery materials for RNA therapeutics.

Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes.

B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T-cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non-Hodgkin's lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose-dependent and time-controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells. Here, the design, synthesis, and biological evaluation of a lipid nanoparticle (LNP) system that can encapsulate mRNA, navigate to the spleen, transfect B lymphocytes, and induce more than 60 pg of protein expression per million B cells within the spleen is described. Importantly, this LNP induces more than 85% of total protein production in the spleen, despite LNPs being observed transiently in the liver and other organs. These results demonstrate that LNP composition alone can be used to modulate the site of protein induction in vivo, highlighting the critical importance of designing and synthesizing new nanomaterials for nucleic acid delivery.

Polymer-Lipid Nanoparticles for Systemic Delivery of mRNA to the Lungs.

Therapeutic nucleic acids hold great promise for the treatment of disease but require vectors for safe and effective delivery. Synthetic nanoparticle vectors composed of poly(ß-amino esters) (PBAEs) and nucleic acids have previously demonstrated potential utility for local delivery applications. To expand this potential utility to include systemic delivery of mRNA, hybrid polymer-lipid nanoformulations for systemic delivery to the lungs were developed. Through coformulation of PBAEs with lipid-polyethylene glycol (PEG), mRNA formulations were developed with increased serum stability and increased in vitro potency. The formulations were capable of functional delivery of mRNA to the lungs after intravenous administration in mice. To our knowledge, this is the first report of the systemic administration of mRNA for delivery to the lungs using degradable polymer-lipid nanoparticles.

Efficacy and immunogenicity of unmodified and pseudouridine-modified mRNA delivered systemically with lipid nanoparticles in vivo.

mRNA has broad potential for treating diseases requiring protein expression. However, mRNA can also induce an immune response with associated toxicity. Replacement of uridine bases with pseudouridine has been postulated to modulate both mRNA immunogenicity and potency. Here, we explore the immune response and activity of lipid nanoparticle-formulated unmodified and pseudouridine-modified mRNAs administered systemically in vivo. Pseudouridine modification to mRNA had no significant effect on lipid nanoparticle physical properties, protein expression in vivo, or mRNA immunogenicity compared to unmodified mRNA when delivered systemically with liver-targeting lipid nanoparticles, but reduced in vitro transfection levels. Indicators of a transient, extracellular innate immune response to mRNA were observed, including neutrophilia, myeloid cell activation, and up-regulation of four serum cytokines. This study provides insight into the immune responses to mRNA lipid nanoparticles, and suggests that pseudouridine modifications may be unnecessary for therapeutic application of mRNA in the liver.

Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery.

Thousands of human diseases could be treated by selectively controlling the expression of specific proteins in vivo. A new series of alkenyl amino alcohol (AAA) ionizable lipid nanoparticles (LNPs) capable of delivering human mRNA with unprecedented levels of in vivo efficacy is demonstrated. This study highlights the importance of utilizing synthesis tools in tandem with biological inspiration to understand and improve nucleic acid delivery in vivo.

Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs.

Intracellular delivery of messenger RNA (mRNA) has the potential to induce protein production for many therapeutic applications. Although lipid nanoparticles have shown considerable promise for the delivery of small interfering RNAs (siRNA), their utility as agents for mRNA delivery has only recently been investigated. The most common siRNA formulations contain four components: an amine-containing lipid or lipid-like material, phospholipid, cholesterol, and lipid-anchored polyethylene glycol, the relative ratios of which can have profound effects on the formulation potency. Here, we develop a generalized strategy to optimize lipid nanoparticle formulations for mRNA delivery to the liver in vivo using Design of Experiment (DOE) methodologies including Definitive Screening and Fractional Factorial Designs. By simultaneously varying lipid ratios and structures, we developed an optimized formulation which increased the potency of erythropoietin-mRNA-loaded C12-200 lipid nanoparticles 7-fold relative to formulations previously used for siRNA delivery. Key features of this optimized formulation were the incorporation of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and increased ionizable lipid:mRNA weight ratios. Interestingly, the optimized lipid nanoparticle formulation did not improve siRNA delivery, indicating differences in optimized formulation parameter design spaces for siRNA and mRNA. We believe the general method described here can accelerate in vivo screening and optimization of nanoparticle formulations with large multidimensional design spaces.

CNS penetration of intrathecal-lumbar idursulfase in the monkey, dog and mouse: implications for neurological outcomes of lysosomal storage disorder.

A major challenge for the treatment of many central nervous system (CNS) disorders is the lack of convenient and effective methods for delivering biological agents to the brain. Mucopolysaccharidosis II (Hunter syndrome) is a rare inherited lysosomal storage disorder resulting from a deficiency of iduronate-2-sulfatase (I2S). I2S is a large, highly glycosylated enzyme. Intravenous administration is not likely to be an effective therapy for disease-related neurological outcomes that require enzyme access to the brain cells, in particular neurons and oligodendrocytes. We demonstrate that intracerebroventricular and lumbar intrathecal administration of recombinant I2S in dogs and nonhuman primates resulted in widespread enzyme distribution in the brain parenchyma, including remarkable deposition in the lysosomes of both neurons and oligodendrocytes. Lumbar intrathecal administration also resulted in enzyme delivery to the spinal cord, whereas little enzyme was detected there after intraventricular administration. Mucopolysaccharidosis II model is available in mice. Lumbar administration of recombinant I2S to enzyme deficient animals reduced the storage of glycosaminoglycans in both superficial and deep brain tissues, with concurrent morphological improvements. The observed patterns of enzyme transport from cerebrospinal fluid to the CNS tissues and the resultant biological activity (a) warrant further investigation of intrathecal delivery of I2S via lumbar catheter as an experimental treatment for the neurological symptoms of Hunter syndrome and (b) may have broader implications for CNS treatment with biopharmaceuticals.

Safety evaluation of chronic intrathecal administration of heparan N-sulfatase in juvenile cynomolgus monkeys.

An intrathecal (IT) formulation of recombinant human heparan N-sulfatase (HNS) is under development for the treatment of the neurological symptoms of mucopolysaccharidosis IIIA (MPS IIIA; Sanfilippo A disease), the defining clinical feature of this disorder. Since the average age of MPS IIIA patients is 4.5 years, the pivotal toxicology studies for HNS were conducted in juvenile cynomolgus monkeys to evaluate the effects on the developing brain. Monkeys were implanted with an IT-lumbar drug delivery device and dosed every other week by slow bolus administration (1.5, 4.5, or 8.3 mg/dose HNS for 6 months; 12 doses), with device and vehicle controls receiving phosphate-buffered saline or vehicle, respectively. Eight animals per group (four/sex) were necropsied at 3 and 6 months (device control group necropsied at 3 months), and eight animals from the vehicle group and the three HNS-dosed groups were necropsied 1 month after the final IT dose. No HNS-related clinical signs or gross central nervous system lesions were observed. Compared with controls, there were cellular infiltrates of slight-to-minimal mean severity in the meninges/perineurium surrounding the brain/spinal cord correlating with transient increases in cerebrospinal fluid (CSF) leukocytes, predominantly eosinophils, which largely resolved 1 month after the final dose. These changes were not associated with any adverse morphological changes in the brain or spinal cord. There appeared to be a dose-related trend toward higher mean CSF HNS levels and in tissue HNS activity levels in the brain, spinal cord, and liver. The no-observed-adverse-effect-level was 8.3 mg/dose given every other week, the highest dose administered.

Safety evaluation of chronic intrathecal administration of idursulfase-IT in cynomolgus monkeys.

Recombinant human idursulfase, an intravenous enzyme replacement therapy indicated for treatment of somatic symptoms of mucopolysaccharidosis II (Hunter syndrome), is anticipated to have minimal benefit for the cognitive impairment associated with the severe phenotype. Because intrathecal (IT) administration of enzyme replacement therapy for other lysosomal enzyme disorders has shown efficacy in animal models, an IT formulation of idursulfase (idursulfase-IT) and a drug-delivery device (subcutaneous port connected to a lumbar IT catheter) were developed for treating central nervous system (CNS) involvement. In this chronic safety study, cynomolgus monkeys were dosed weekly with IV idursulfase (0.5 mg/kg) and every four weeks with idursulfase-IT (3, 30, and 100 mg) for six months, with device and vehicle controls treated similarly (n = 6, all groups). Necropsies were performed twenty-four hours post-final IT dose or after a recovery period (four weeks post-final dose in vehicle-control, 3 mg, and 100 mg IT groups: n = 6). No clinical signs or gross central nervous system lesions were observed. Compared to controls, more pronounced cellular infiltrates in brain and spinal cord meninges were noted, which largely resolved after the recovery period. Central nervous sytem levels of idursulfase-IT were dose dependent, as determined by enzyme activity and immunohistochemistry. The no-observed-adverse-effect level of idursulfase-IT was 100 mg.

Erythropoietin produced in a human cell line (Dynepo) has significant differences in glycosylation compared with erythropoietins produced in CHO cell lines.

Recombinant human erythropoietin has been used to treat anemia associated with chronic renal disease. This paper provides a comprehensive comparative analysis of Dynepo and three other commercial erythropoiesis stimulating agents, Eprex, NeoRecormon and Aranesp. We found significant differences in the type, levels and amount of O-acetylation of sialic acids. Sialic acids and O-acetylation present provide protection from clearance from circulation. Aranesp had up to six O-acetyl groups attached to the sialic acids. Eprex and NeoRecormon had only minor amounts of O-acetylation while Dynepo had none. Dynepo had no Neu5Gc, which is potentially immunogenic for humans. Dynepo contained the least amount of disialylated and Aranesp the highest amount of tetrasialylated glycans. NeoRecormon and Eprex contained more trisialylated, but less tetrasialylated glycans than Dynepo and Aranesp. Dynepo had the highest amount of tetraantennary glycans and the lowest amounts of triantennary glycans with a ß1-6-GlcNAc linkage. All the samples contained poly-N-acetyl-lactosamine repeats with Dynepo having the least. The major N-acetyl-lactosamine extensions in Dynepo and Aranesp were on biantennary glycans, whereas in NeoRecomon and Eprex they were on triantennary glycans. The sLe(x) epitope was only detected in Dynepo.

Human Sulfatase 2 inhibits in vivo tumor growth of MDA-MB-231 human breast cancer xenografts.

BACKGROUND: Extracellular human sulfatases modulate growth factor signaling by alteration of the heparin/heparan sulfate proteoglycan (HSPG) 6-O-sulfation state. HSPGs bind to numerous growth factor ligands including fibroblast growth factors (FGF), epidermal growth factors (EGF), and vascular endothelial growth factors (VEGF), and are critically important in the context of cancer cell growth, invasion, and metastasis. We hypothesized that sulfatase activity in the tumor microenvironment would regulate tumor growth in vivo. METHODS: We established a model of stable expression of sulfatases in the human breast cancer cell line MDA-MB-231 and purified recombinant human Sulfatase 2 (rhSulf2) for exogenous administration. In vitro studies were performed to measure effects on breast cancer cell invasion and proliferation, and groups were statistically compared using Student's t-test. The effects of hSulf2 on tumor progression were tested using in vivo xenografts with two methods. First, MDA-MB-231 cells stably expressing hSulf1, hSulf2, or both hSulf1/hSulf2 were grown as xenografts and the resulting tumor growth and vascularization was compared to controls. Secondly, wild type MDA-MB-231 xenografts were treated by short-term intratumoral injection with rhSulf2 or vehicle during tumor growth. Ultrasound analysis was also used to complement caliper measurement to monitor tumor growth. In vivo studies were statistically analyzed using Student's t test. RESULTS: In vitro, stable expression of hSulf2 or administration of rhSulf2 in breast cancer cells decreased cell proliferation and invasion, corresponding to an inhibition of ERK activation. Stable expression of the sulfatases in xenografts significantly suppressed tumor growth, with complete regression of tumors expressing both hSulf1 and hSulf2 and significantly smaller tumor volumes in groups expressing hSulf1 or hSulf2 compared to control xenografts. Despite significant suppression of tumor volume, sulfatases did not affect vascular density within the tumors. By contrast, transient exogenous treatment of MDA-MB-231 xenografts with rhSulf2 was not sufficient to inhibit or reverse tumor growth. CONCLUSION: These data indicate that in vivo progression of human breast cancer xenografts can be inhibited with sulfatase expression, and therapeutic effect requires constant delivery at the tumor site. Our results support a direct effect of sulfatases on tumor growth or invasion, rather than an effect in the stromal compartment.

Recombinant human complement component C2 produced in a human cell line restores the classical complement pathway activity in-vitro: an alternative treatment for C2 deficiency diseases.

BACKGROUND: Complement C2 deficiency is the most common genetically determined complete complement deficiency and is associated with a number of diseases. Most prominent are the associations with recurrent serious infections in young children and the development of systemic lupus erythematosus (SLE) in adults. The links with these diseases reflect the important role complement C2 plays in both innate immunity and immune tolerance. Infusions with normal fresh frozen plasma for the treatment of associated disease have demonstrated therapeutic effects but so far protein replacement therapy has not been evaluated. RESULTS: Human complement C2 was cloned and expressed in a mammalian cell line. The purity of recombinant human C2 (rhC2) was greater than 95% and it was characterized for stability and activity. It was sensitive to C1s cleavage and restored classical complement pathway activity in C2-deficient serum both in a complement activation ELISA and a hemolytic assay. Furthermore, rhC2 could increase C3 fragment deposition on the human pathogen Streptococcus pneumoniae in C2-deficient serum to levels equal to those with normal serum. CONCLUSIONS: Taken together these data suggest that recombinant human C2 can restore classical complement pathway activity and may serve as a potential therapeutic for recurring bacterial infections or SLE in C2-deficient patients.