Vancouver, British Columbia, February 21, 2017 –

The mission of Precision NanoSystems (PNI) is to empower the creation and development of innovative nanomedicines. The motto “Create transformative medicines” was officially adopted in January 2017, but has been embodied throughout PNI’s history. The fruit of this commitment is encompassed in the work of innovators who have adopted PNI’s lipid nanoparticle (LNP) transfection technology and the NanoAssemblr™ platform.  In 2016, 27 peer-reviewed papers and reviews featuring PNI technology were published, which continues the trend of year-over-year doubling. This surge in productivity occurred across a broad cross-section of application areas, namely functional genomics, neuroscience, gene and cell therapy, oncology, and drug delivery & formulation. Highlights from each of these areas are reviewed here.

Functional genomics

While most users of PNI technology are drug formulation scientists, the utility of the NanoAssemblr platform for making lipid nanoparticles that are highly potent transfection agents has not been lost on disease researchers. For example, researchers from the University of Manitoba discovered a new molecular mechanism of insulin-stimulated glucose uptake in Clone 9 cells. This is significant because Clone 9 cells are commonly used to study insulin signalling and glucose uptake in the liver and this discovery defies the established understanding. Their findings were reported in Biochemical and Biophysical Research Communications. In early studies on Clone9 cells, several isoforms of the facilitated glucose transporter (GLUT) were screened by Northern blot. The GLUT3 isoform was not screened for and GLUT1 mRNA was the only isoform thought to be expressed. From there, numerous reports suggested that GLUT1 is the only glucose transporter expressed in Clone9 cells. Using modern techniques, the study authors profiled GLUT1-GLUT5 expression in Clone 9 cells and were able to detect GLUT1 and GLUT3 mRNA while only GLUT3 protein was present. These findings were confirmed by using PNI’s Test9™ transfection nanoparticles to deliver short interfering RNA (siRNA) to mediate knockdown of GLUT3 via the RNA interference (RNAi) pathway. GLUT3 knockdown abolished glucose uptake and the results indicated that GLUT3 – and not GLUT1 – is largely responsible for glucose transport. This discovery has far reaching implications on studies that used Clone 9 cells to study GLUT1 mediated glucose uptake. It also suggests opportunities to reinterpret past results, revisit other assumptions armed with modern techniques, and to study the regulation of GLUT3 in this cell line. Effective gene manipulation, particularly with RNA-LNPs is an essential tool in all of those endeavours.

Neuroscience

In this category, there are papers that overlap with functional genomics and gene therapy.  Nevertheless, neuroscience offers unique challenges that can be addressed with PNI technology. In particular, the challenges to transfecting neurons have posed barriers to translating genomics insights into genetic treatments for diseases of the brain. Efficient and practical transfection methods are needed to functionally test these insights in relevant cells and, at later stages of development, deliver therapeutic nucleic acids. Two papers published in 2016 describe compelling proof-of-concept studies that address functional screening and in vivo mRNA replacement therapy in neuronal cell types. In both cases, lipid nanoparticles (LNPs) were used to encapsulate and deliver RNA to neurons.

To address challenges to functional screening, researchers at the Weizmann Institute in Israel combined robotic fluid handling and microscopy with PNI’s Neuro9™ transfection technology to create a platform for screening the effects of supressing or expressing genes on observable cell phenotypes. In a paper published in Neuromethods, they described their process for harvesting primary motor neurons extracted from the spinal cords of embryonic mice and culturing them in 384-well format. The cells were then transfected with microRNA mimics and their phenotypes observed by automated microscopy. The potency and lack of observable toxicity allow Neuro9 to overcome challenges associated with other conventional methods. Furthermore, because Neuro9 can be applied directly to culture without disturbing the cells, they are ideal for workflow integration with automated liquid handling systems. The authors suggest this system can be used to understand microRNA function in a variety of neuropathies. In principle, the platform can be used for numerous RNA screening applications. This is an important development for functional genomics, target validation and preclinical development of genetic medicines for neurodegenerative diseases.

Also pivotal to developing genetic interventions to disorders of the central nervous system (CNS), is demonstrating in vivo delivery of nucleic acids to neurons.  While publications in 2015 demonstrated RNAi-mediated gene knockdown following cortical injection of siRNA-LNPs, in a new development for 2016, researchers at Pfizer’s rare disease research unit demonstrated spinal injection of messenger RNA (mRNA) LNPs and expression of exogenous proteins in rats. The paper, which appeared in the February 2016 edition of Scientific Reports, suggests that mRNA replacement therapy can be used to treat diseases where mutations cause a loss of protein function. mRNA is particularly challenging to deliver compared to shorter siRNA and DNA because mRNA is inherently less stable and biological mechanisms have evolved to degrade mRNA and limit its life time. As a model disease, the study authors chose Friedrich’s Ataxia, where diminished levels of the protein frataxin causes symptoms of neurodegeneration and pathology in neurons. Codon optimized mRNA encoding human frataxin was delivered by lumbar injections of mRNA-LNPs that were made on the NanoAssemblr Benchtop. Expression of the protein was detected in dorsal root ganglia near the injection site, but was not elsewhere in the CNS. The exogenous human frataxin was almost completely processed into the mature form, which only takes place in the mitochondrion where the protein performs its function. This suggests the protein is bioavailable and could be functioning as intended.  The exogenous protein was found at three-times the abundance of mouse frataxin. These findings represent the first demonstration of exogenous protein expression in dorsal root ganglia following in vivo mRNA delivery, and mark a milestone towards the development of future nucleic acid therapies for tissues of the CNS.

These demonstrations of in vitro functional genomic screening and in vivo therapies for neurons serve as proof of concept for several stages of early therapeutic development. Considering the challenges of neuronal transfection by conventional means, these developments have immense impact in the field and highlight the utility of Neuro9 LNPs in disease research and therapeutic development.

Gene and cell therapy

An essential step in gene therapy is delivering nucleic acids across cell membranes so they can mediate gene knockdown through the RNAi pathway, or the expression of exogenous proteins. Of course, gene-editing techniques have great potential for therapeutic applications and CRISPR/Cas9 in particular has made headlines in the press. Given the ability of LNP technology to deliver nucleic acids, there is interest in using LNPs as non-viral alternatives for delivering CRISPR components into cells in the form of mRNA encoding Cas9 and multiple short RNA guide strands. LNPs have the potential to overcome some of the limitations of viral vectors.  Two papers from 2016 that highlight these topics are notable.

The first paper of note focuses on the cellular mechanism governing uptake of LNPs and release of the nucleic acid payload.   LNPs are taken up by receptor mediated endocytosis, and are designed to release their payload in response to acidification of the endosome. Based on previous findings that the Niemann-Pick type C1 (NPC1) knockout cells showed enhanced silencing when treated with siRNA LNPs, researchers from the Cullis Lab at the University of British Columbia hypothesized that pharmacological inhibition of NPC1 could enhance the release of siRNA into the cytoplasm. In their paper published in Molecular Therapy, the researchers use a pharmacological agent to inhibit NPC1, which was previously found to inhibit Ebola virus infection. NPC1 is a membrane protein involved in recycling cholesterol from low-density lipoproteins taken up by receptor-mediated endocytosis. Inhibiting NPC1 had the effect of trapping the Ebola virus in the endosome. For LNPs, which are taken up in the endocytic pathway, LNPs spend more time in the endosome providing a greater opportunity to trigger payload release. The researchers report a reduction in the siRNA dose required to knockdown a gene when the NPC1 inhibitor was used, but the magnitude was found to vary by cell type. Nevertheless these findings suggest a way to reduce RNA doses in LNP therapeutics.

In the second key paper,  researchers from the University of Texas South Western, describe the use of lipid nanoparticles for delivering CRISPR components for gene editing. This constitutes the first demonstration of non-viral co-delivery of CRISPR/Cas components and subsequent editing both in vitro and in vivo.  CRISPR gene editing is a powerful tool that acts like a find-and-replace function for genomic DNA. As a therapeutic, it has the potential to fix the root cause of many genetic ailments. There remain however, many challenges to overcome, not the least of which is delivering the molecular machinery in a pharmaceutically acceptable way. For CRISPR to work, both the Cas9 protein, which cleaves DNA, and the guide RNA (sgRNA), which imparts specificity to the cleavage site, must be delivered, be simultaneously bioavailable and unite in the cytoplasm. LNP co-delivery of both the sgRNA and Cas9-mRNA in the same formulation has several advantages over viral vectors.  First, viral vectors have limitations to the length of nucleic acid sequences that can be packed into the viral genome and having both Cas9 and guide sequences would exceed this limit. Second, co-encapsulation of the mRNA and guide leads to simultaneous delivery, which ensures each cell will receive both molecules. This avoids the pharmacological complexity of delivering separate vehicles. Finally, nucleic acid LNPs can be prepared in minutes on the NanoAssemblr Benchtop and function in vitro and in vivo, while viral vectors take days to prepare, which is prohibitively laborious for in vitro screening applications.

In both of these studies, these groups used their own proprietary lipid formulations, which they formulated into LNPs on the NanoAssemblr Benchtop instrument.

Oncology

In 2016, cancer therapeutics research continued to be a strong area of interest among NanoAssemblr users with siRNA-based cancer treatments accounted for the majority of oncology related publications.  Prominent among these are antibody or peptide conjugated LNPs that promote uptake by specific cells. The Peer Lab at Tel Aviv University reported in the Proceedings of the National Academy of Sciences, an antibody conjugated siRNA-LNP that significantly improved survival of mice bearing mantle cell lymphoma. siRNA against cyclin D1 – a protein overexpressed in mantle cell lymphoma – was encapsulated into LNPs containing chemically active lipids using the NanoAssemblr Benchtop. Antibodies known to bind to surface markers of the diseased cells were conjugated to the chemically active groups on the surface of the LNPs.  Antibody targeting allowed the authors to administer the LNP in the blood stream of tumour-bearing mice and observe siRNA transfection in the target cells. Without the antibody, these particles would be expected to accumulate in the liver, where they would transfect liver cells. Targeted LNPs were found to transfect the intended cells with specificity compared to non-targeted particles. Further, in controlled experiments, targeted LNPs were found to extended survival by a statistically significant period compared to control groups either treated with non-targeting LNPs or untreated.

In another targeted LNP study, the Cullis Lab at the University of British Columbia have reported in the journal Molecular Therapy, a ligand-conjugated LNP that targets the prostate specific membrane antigen (PSMA) overexpressed in prostate cancer cells but not in healthy tissue. Targeting is desirable because biodistribution of many LNP formulations do not favour prostate tumour accumulation, and thus higher doses of siRNA are required. The PSMA-targeting ligand Glu-urea-lys-PEG-lipid was synthesized then combined with other LNP-forming lipids.  Using the NanoAssemblr Benchtop, the lipids were mixed with siRNA against the androgen receptor to form the ligand-conjugated siRNA LNPs. This method contrasts with the previous example where LNPs were formed prior to ligand conjugation. The Cullis group also included other formulation refinements that led to longer circulation half-life and improved efficacy. In mice bearing human prostate tumour xenografts, PSMA-targeted LNPs were found to be more effective than nontargeted LNPs with respect to reducing malignant cell proliferation, reducing serum levels of the prostate specific antigen (PSA) and reducing mRNA levels of both PSA and the target androgen receptor.

In spite of continued debate about the merits of targeting ligands in nanomedicine, these findings suggest that rational formulation decisions aimed at improving circulation times will help targeting ligands reach their desired targets where they can mediate extended interactions with target biomarkers. Other approaches such as non-systemic injections or tuning of size and surface chemistry are also effective ways of controlling accumulation in specific tissues. In 2016, there were two notable publications featuring PNI technology that used untargeted LNPs for cancer therapy. The first, appeared in the January issue of PNAS and featured a dendrimer-based nanoparticle for treating liver cancer. It’s widely understood that PEGylated particles between 50-100 nm accumulate readily in liver following systemic injection. The second, published in Molecular Therapy-Nucleic Acids featured a lipid formulation proprietary to Arcturus called LUNAR. This study followed up on the group’s prior in vitro work with in vivo data demonstrating that siRNA mediated knockdown of Cdk16 inhibits tumour growth in tumour xenografts derived from colorectal cancer and melanoma cell lines.

There were also several reviews published in 2016 covering topics related to preclinical development of nanomedicines for oncology. Of particular interest, is a review of developments in the Cellax drug delivery platform.  Cellax consists of a carboxymethyl cellulose polymer backbone to which is grafted small molecule chemotherapies and other functional pendant groups. These polymers spontaneously condense into nanoparticles in aqueous environments, and the researchers have been using the NanoAssemblr Benchtop to improve reproducibility and polydispersity, which translates into improved size control and biodistribution. The review underscores the impressive preclinical development of the platform including a 2015 paper describing how Cellax nanoparticles below 30nm in diameter improved passive accumulation in tumours relative to other tissues, which greatly improved survival in animal studies. The Peer Lab also published a review of LNP-based RNAi therapeutics for leukocytes.

The following section on drug delivery and formulation is also relevant to oncology due to the interest in encapsulating hydrophobic drugs like cytotoxic chemotherapeutics to alter biodistribution and mitigate off-target toxicity.

Drug Delivery and Formulation

This category covers papers that focus on formulation science, chemistry and characterization or nanomedicines. Applications for these innovations are broad and can benefit delivery of small molecules and nucleic acids alike. Implied is an overlap with oncology applications where nanomedicine formulations are being explored to improve the efficacy and reduce off-target toxicity of insoluble chemotherapeutic small molecules. This category also covers a broad range of carrier materials including polymers, dendrimers, and various lipids and surfactants. Two notable studies are highlighted.

First, the Dong lab at Ohio State University presented a study on optimizing a lipid-like compound for mRNA delivery in the journal Scientific Reports. mRNA delivery can be more challenging than siRNA delivery because mRNA is less stable and is often more than 100-fold larger, which reduces the molarity of the delivered cargo. The researchers synthesized lipid-like molecules with minor structural and chemical variations and tested formulations with other helper lipids to identify the formulation that produced the greatest expression levels of the encoded protein. Their lead formulation exhibited 10-fold greater delivery efficiency that other formulations. Using the NanoAssemblr Benchtop, they were able to formulate the lead candidate for in vivo use where they found, unexpectedly, that their LNPs accumulated preferentially in the spleen following systemic and intraparitoneal injection. Other PEGylated lipid-like nanoparticles have been shown to accumulate in the liver. This finding may lead to future treatments to target the spleen, for example to access immune cells that are abundant there.

Second, the Perrie Lab at Strathclyde University demonstrated for the first time, liposome precipitation with simultaneous loading of both a hydrophilic and a hydrophobic drug. The one-step formulation and loading of two drugs will significantly reduce the complexity of scale up. High loading efficiencies of up to 25% and 42% were reported for the passive encapsulation of hydrophilic and hydrophobic drugs respectively. Interestingly, the drug release was found to occur more quickly with the dual-loaded liposomes than for single-drug formulations. Importantly, drug-loading efficiencies remained the same for the dual-loaded liposome as for loading each drug individually. These results are promising for development of combination therapies that can potentially have synergistic effects.

Other studies of note include: 1) a method to rapidly quantify lipid concentrations in liposomes, which can be valuable in assessing product quality and recovery, 2) a report of NanoAssemblr manufacture of monodisperse micron-sized lipid particles with cochleate morphology, and 3) Formulation of non-ionic surfactant vesicles (NISVs) on the NanoAssemblr Benchtop.

This category of publications covers a variety of payloads, carrier materials, and applications.  The variety of formulations reported by Benchtop users underscores the versatility of the platform and the creativity of the innovators who have adopted it.

Trends

In 2016, NanoAssemblr and transfection kit users combined to produce a strong body of publications across a broad range of disciplines that span disease research, early discovery and various stages of preclinical development. Several key trends emerge from this body of work.

First, building upon seminal work demonstrating LNP transfection in neurons, the launch of Neuro9 LNP transfection agents is pivotal to understanding the genetic basis of neurodegenerative diseases and to creating new therapeutics. In 2016 delivery of both siRNA and mRNA to neuronal cells in vitro and in vivo was reported. These findings challenge the widely held belief that neurons are difficult to transfect and manipulate genetically. Highly efficacious and ostensibly non-toxic transfection lowers barriers to understanding the molecular mechanisms of neural diseases and allows for new experiments and treatments to be imagined. This foreshadows a growth in discoveries in neuroscience and preclinical development of new treatments for neurodegenerative diseases.

This interest in mRNA delivery to neurons is reflective of a broader interest in mRNA delivery to other cell types. This is indicative that the community is overcoming the challenges of mRNA delivery through the use of lipid-based formulations. The ability to express exogenous proteins is a natural complement to siRNA mediated gene knockdown. This opens the door to gene replacement for research and therapeutic use, but also to potential immuno-oncology applications where cancer antigens can be delivered in mRNA format to immune cells, and of course the delivery of gene editing nucleases like Cas9. Indeed, studies led by the Peer Lab have demonstrated successful transfection of immune cells by antibody targeted LNPs, while efforts by the Dong Lab have identified synthetic lipid-like compounds the preferentially accumulate in the leukocyte-rich spleen. Taken together, these are indicative of a trending interest in co-opting immune cells to treat cancer and other diseases.

Developments in targeting LNPs using conjugated ligands is also a growing area of interest. Integral to this is a growing understanding of how to rationally improve circulation half-life and avoid sequestration in undesired tissues, which is a necessary step to allow the targeting ligands to direct particle accumulation. Closely related to this is the growing understanding of how size, surface charge, and other physical and chemical properties affect biodistribution of nanomedicines. Research interest in these areas is certain to trend upwards in 2017.

Another possible pattern emerging from these publications is a trend towards combination therapies. While the Cullis Lab demonstrated that carefully selected pharmaceutical small molecules can improve the potency of siRNA transfection, work by the Perrie lab demonstrated a means by which to combine hydrophobic and hydrophilic active molecules in a single step, which can be generalized to deliver numerous combinations of active ingredients.

Overall, the community has combined to produce an impressive body of work in 2016. If this trend continues, 2017 will turn out to be another record year. This productivity is a reflection of not only workflow improvements, but vitally, the productivity gained from entrusting a crucial portion of the process to PNI technology so that researchers are free to focus on aspects of their work that most ignites their interest.