Delivery Of Nucleic Acid-based Therapeutics Research Articles

Intranasal delivery: An attractive route for the administration of nucleic acid based therapeutics for CNS disorders.

The etiologies of several cardiovascular, inflammatory, neurological, hereditary disorders, cancer, and infectious diseases have implicated changes in the genetic set up or genetic mutations as the root cause. Nucleic acid based therapeutics (NBTs) is a new class of biologics that are known to regulate gene expression at the transcriptional and post-transcriptional level. The NBTs include oligonucleotides, nucleosides, antisense RNA, small interfering RNAs, micro RNA etc. In recent times, this new category of biologics has found enormous potential in the management of cardiovascular, inflammatory, neurological disorders, cancer, infectious diseases and organ transplantation. However, the delivery of NBTs is highly challenging in terms of target specificity (intracellular delivery), mononuclear phagocyte system uptake, stability and biodistribution. Additionally, management of the above mentioned disorders require regular and intrusive therapy making non-invasive routes preferable in comparison to invasive routes like parenteral. The nasal route is garnering focus in delivery of NBTs to the brain in the management of several CNS disorders due to the associated merits such as non-invasiveness, possibility of chronic delivery, improved patient compliance, avoidance of hepatic and gastrointestinal metabolism as well as ability to bypass the BBB. Hence in recent times, this route has been sought by the reserachers as an alternative to parenteral therapy for the delivery of several NBTs. This review shall focus on an array of NBTs delivered through nasal route, their challenges, applications and opportunities. The novel delivery systems for incorporating NBTs; their targeting strategies shall be critically reviewed. The challenges towards regulatory approvals and commercialization shall also be discussed at large. Comparison of learnings derived from the success and barriers in nasal delivery of NBTs will help in identification of futuristic opportunities for their translation from bench to bedside.

Smart Nanocarriers for the Delivery of Nucleic Acid-Based Therapeutics: A Comprehensive Review.

Nucleic acid-based therapies are promising therapeutics for the treatment of several systemic disorders, and they offer an exciting opportunity to address emerging biological challenges. The scope of nucleic acid-based therapeutics in the treatment of multiple disease states including cancers has been widened by recent progress in Ribonucleic acids (RNA) biology. However, cascades of systemic and intracellular barriers, including rapid degradation, renal clearance, and poor cellular uptake, hinder the clinical effectiveness of nucleic acid-based therapies. These barriers can be circumvented by utilizing advanced smart nanocarriers that efficiently deliver and release the encapsulated nucleic acids into the target tissues. This review describes the current status of clinical trials on nucleic acid-based therapeutics and highlights representative examples that provide an overview on the current and emerging trends in nucleic acid-based therapies. A better understanding of the design of advanced nanocarriers is essential to promote the translation of therapeutic nucleic acids into a clinical reality.

Design of Peptidomimetic Functionalized Cholesterol Based Lipid Nanoparticles for Efficient Delivery of Therapeutic Nucleic Acids.

Lipid nanoparticles (LNP) are the most potent carriers for the delivery of nucleic acid-based therapeutics. The first FDA approved a short interfering RNA (siRNA) drug that uses a cationic LNP system for the delivery of siRNA against human transthyretin (hTTR). However, preparation of such LNP involves tedious multi-step synthesis with relatively low yields. In the present study, we synthesized cationic peptidomimetic functionalized cholesterol (denote Chorn) in straightforward chemical approaches with high yield. When formulated with helper lipids, Chorn LNPs complexed with siRNA to form nanoparticles with an average diameter of 150 nm to 200 nm. Chorn LNP mediated transfection of a green fluorescence protein (GFP) expressing plasmid resulted in 60% GFP positive cells. Moreover, Chorn LNP delivered siRNA against polo-like kinase 1 (Plk1), a disease related gene in cancer cells and efficiently suppressed the expression of the gene, resulting in significant morphological changes in the cell nuclei. Our data suggested that cholesterol based cationic LNP, prepared through a robust chemical strategy, may provide a promising siRNA delivery system.

Self-assembling asymmetric peptide-dendrimer micelles \u2013 a platform for effective and versatile in vitro nucleic acid delivery

Despite advancements in the development of high generation cationic-dendrimer systems for delivery of nucleic acid-based therapeutics, commercially available chemical agents suffer from major drawbacks such as cytotoxicity while being laborious and costly to synthesize. To overcome the aforementioned limitations, low-generation cationic peptide asymmetric dendrimers with side arm lipid (cholic and decanoic acid) conjugation were designed, synthesized and systematically screened for their ability to self-assemble into micelles using dynamic light scattering. Cytotoxicity profiling revealed that our entire asymmetric peptide dendrimer library when trialled alone, or as asymmetric dendrimer micelle-nucleic acid complexes, were non-cytotoxic across a broad concentration range. Further, the delivery efficiency of asymmetric peptide dendrimers in H-4-II-E (rat hepatoma), H2K (mdx mouse myoblast), and DAOY (human medulloblastoma) cells demonstrated that cholic acid-conjugated asymmetric dendrimers possess far superior delivery efficiency when compared to the commercial standards, Lipofectamine 2000 or Lipofectin®.

621. Widespread Gene Delivery to the Nonhuman Primate Brain for the Treatment of Huntington's Disease

Huntington disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG-trinucleotide repeat expansion in a coding exon of a single allele in the HTT locus. In HD, the resulting polyglutamine (polyQ) expansion confers a toxic gain-of-function to the mutant huntingtin protein (mHTT). Reduction of expression of mHTT using gene silencing by RNA interference (RNAi) may confer transformative disease modifying therapeutic approach for HD. Adeno associated vectors (AAV) provide an ideal delivery system for nucleic acid therapeutics and have the potential to allow for long lasting and continuous expression of these huntingtin lowering RNAi in the brain. Despite this promise, global delivery of AAV to the adult brain remains an elusive goal. Furthermore, the appropriate brain areas to target for achieving transformative therapeutic benefit in HD patients remain to be defined. Postmortem analyses of HD patient brains reveal extensive medium spiny neuronal loss in the striatum, in addition to loss of pyramidal neurons in the cerebral cortex and hippocampus. Recent studies in rodent models suggest that simultaneous targeting of striatum and cortex is more efficacious than targeting either individually. Thus, available evidence suggests that delivery of Htt-lowering therapeutics to both striatal and cortical regions may provide optimal therapeutic efficacy. The current study demonstrate for the first time the successful use of an AAV targeting strategy that leads to viral transduction in key brain areas considered to be important for HD pathology. The study compared the efficiency of transduction of AAV1 and AAV2 vectors in the rhesus monkey brain following intra-striatal injection. Both vectors encoded green fluorescent protein (GFP) under control of a hybrid CMV enhancer/chicken beta-actin promoter. One month following injection, brains were analyzed for distribution of GFP-positive cells. We found that the AAV1 vector provided extensive delivery to the majority of the primate striatum, and additionally transduced large numbers of cells within the cerebral cortex, thalamus, and hippocampus. In summary, the data suggest that intrastriatal delivery maybe sufficient for the delivery of nucleic acid-based therapeutics to multiple areas of the human brain relevant in HD.

Self-assembled ternary complexes stabilized with hyaluronic acid-green tea catechin conjugates for targeted gene delivery

Nanosized polyelectrolyte complexes are attractive delivery vehicles for the transfer of therapeutic genes to diseased cells. Here we report the application of self-assembled ternary complexes constructed with plasmid DNA, branched polyethylenimine and hyaluronic acid-green tea catechin conjugates for targeted gene delivery. These conjugates not only stabilize plasmid DNA/polyethylenimine complexes via the strong DNA-binding affinity of green tea catechin, but also facilitate their transport into CD44-overexpressing cells via receptor-mediated endocytosis. The hydrodynamic size, surface charge and physical stability of the complexes are characterized. We demonstrate that the stabilized ternary complexes display enhanced resistance to nuclease attack and polyanion-induced dissociation. Moreover, the ternary complexes can efficiently transfect the difficult-to-transfect HCT-116 colon cancer cell line even in serum-supplemented media due to their enhanced stability and CD44-targeting ability. Confocal microscopic analysis demonstrates that the stabilized ternary complexes are able to promote the nuclear transport of plasmid DNA more effectively than binary complexes and hyaluronic acid-coated ternary complexes. The present study suggests that the ternary complexes stabilized with hyaluronic acid-green tea catechin conjugates can be widely utilized for CD44-targeted delivery of nucleic acid-based therapeutics.

Patenting drug delivery technology: tips for formulating practical and effective patent portfolio.

Pharmaceutical Patent AnalystVol. 5, No. 2 CommentaryPatenting drug delivery technology: tips for formulating practical and effective patent portfolioTakeshi S KomataniTakeshi S Komatani*Author for correspondence: E-mail Address: tskomatani@shupat.gr.jp SHUSAKU·YAMAMOTO, Osaka, JapanSearch for more papers by this authorPublished Online:18 Feb 2016https://doi.org/10.4155/ppa.15.42AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail View articleKeywords: active pharmaceutical ingredientdrug delivery systemintellectual propertypatentpatent term extensionportfoliosupplementary protection certificatePapers of special note have been highlighted as: •• of considerable interestReferences1 Coelho JF, Ferreira PC, Alves P et al. Drug delivery systems: advanced technologies potentially applicable in personalized treatments. EPMA J. 1(1), 164–209 (2010).Crossref, Medline, Google Scholar2 Mastrobattista E, Hennink WE, Schiffelers RM. Delivery of nucleic acids. Pharm. Res. 24(8), 1561–1563 (2007).Crossref, Medline, CAS, Google Scholar3 Xu L, Anchordoquy T. Drug delivery trends in clinical trials and translational medicine: challenges and opportunities in the delivery of nucleic acid-based therapeutics. J. Pharm. Sci. 100(1), 38–52 (2011).Crossref, Medline, CAS, Google Scholar4 US Code: Title 35 (35 USC) Sec. 101.Google Scholar5 Japanese Patent Act. Article 29(1), main paragraph.Google Scholar6 European Patent Convention Article 53(c).Google Scholar7 European Paten Office. www.epo.org/law-practice/case-law-appeals/recent/t080234eu1.html.Google Scholar8 The Commissioner of Japan Patent Office v. Takeda Pharmaceutical Inc. The Supreme Court of Japan, Heisei 21 (Gyo-Hi) no.324–326, 28 April 2012 (2012). ••These decisions, directed to the patent term extension based on a drug delivery system patent, set out the Japanese Supreme Court's position on what is necessary for obtaining patent term extensions, based on the premise that the drug delivery system patent is ‘valid’.Google Scholar9 Komatani TS. Patent term extension in Japan: challenges still lay ahead even after the 2011 revision. Pharma. Pat. Anal. 2(1), 63–76 (2012).Link, Google Scholar10 Japan Patent Attorneys Association. www.jpaa.or.jp/english/court_decisions/6-PacifCapsuleCase.pdf.Google Scholar11 Regulation (EC) no. 469/2009 of the European Parliament and of the Council of 6 May 2009 concerning the supplementary protection certificate for medicinal products.Google Scholar12 Massachusetts Institute of Technology. Case C-431/04 (2006). http://curia.europa.eu/juris/showPdf.jsf?text=&docid=56613&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=642888.Google Scholar13 Neurim Pharmaceuticals Ltd. v. The Comptroller General of Patents. Court of Justice of the European Union, C-130/11; 19 July 2012 (2012).Google Scholar14 D'Oca G, Sklan A, Komatani TS. Cases update. Pharm. Pat. Anal. 1(4), 372–374 (2012).Google Scholar15 MPEP section 2750. www.uspto.gov/web/offices/pac/mpep/s2750.html.Google Scholar16 JRC Science & Policy Report, Farid, Reasonable & Non-Discriminatory (FRAND) Licensing Terms (2015). http://is.jrc.ec.europa.eu/pages/ISG/EURIPIDIS/documents/05.FRANDreport.pdf.Google Scholar17 The White House. www.whitehouse.gov/the-press-office/2015/01/20/remarks-president-barack-obama-prepared-delivery-state-union-address.Google ScholarFiguresReferencesRelatedDetails Vol. 5, No. 2 Follow us on social media for the latest updates Metrics Downloaded 64 times History Published online 18 February 2016 Published in print March 2016 Information© Future Science LtdKeywordsactive pharmaceutical ingredientdrug delivery systemintellectual propertypatentpatent term extensionportfoliosupplementary protection certificateDisclaimerThis commentary describes the opinions and observations of the author as a Japanese patent attorney and does not necessarily represent the viewpoints of the author's employers or author's firm. By its nature, the subject article provides the author's general opinion, and necessarily limited discussion of various topics; it does not purport to give specific legal advice or a substitute for legal counsel. As legal advice must be tailored to the specific circumstances of each case, nothing provided herein should be used as a substitute for advice of competent counsel. The author and the author's firm assume no liability for the use or interpretation of information contained herein. The author and the author's firm expressly disclaim all liability in respect of anything done or omitted to be done wholly or partly in reliance of this material.Financial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

Amphiphilic polyallylamine based polymeric micelles for siRNA delivery to the gastrointestinal tract: In vitro investigations

Targeting the gastrointestinal (GI) tract represents a promising strategy for local or systemic delivery of nucleic acid-based therapeutics. The development of a nano-carrier for siRNA delivery via the GI tract would enable localised therapy for a range of gastrointestinal diseases. Previously, nano-sized polymeric micelles (PM) formed by a variety of amphiphilic poly(allylamine) (PAA) derivatives have shown potential for oral delivery of hydrophobic drugs and bioactive peptides. The aim of this study was to evaluate the ability of these amphiphilic PAA-based PM for siRNA delivery via the GI tract. The physicochemical characteristics of PAA·siRNA transfection complexes and their biological efficacy in vitro were investigated. Physicochemical profiles demonstrated that PAAs and siRNA self-assembled into complexes with nano-sized diameters (150-300 nm) and cationic surface charge (+ 20 to 30 mV). The PAA·siRNA complexes were stable in the presence of salt solutions and simulated gastric/intestinal fluids. In undifferentiated Caco-2 cells, PAA·siRNA complexes achieved up to 35% cellular uptake, with successful siRNA release from endosomes/lysosomes and significant luciferase gene knockdown. These results highlight the potential of these nano-sized PM for siRNA oral delivery via the GI tract for treatment of gastrointestinal diseases.

Oral Delivery of Nucleic acid-based Therapeutics

Oral Delivery of Nucleic acid-based Therapeutics

Tuning Physical Properties of Nanocomplexes through Microfluidics-Assisted Confinement

The future of genetic medicine hinges on successful intracellular delivery of nucleic acid-based therapeutics. While significant effort has concentrated on developing nanocarriers to improve the delivery aspects, scant attention has been paid to the synthetic process of poorly controlled nanocomplex formation. Proposed here is a reliable system to better control the complexation process, and thus the physical properties of the nanocomplexes, through microfluidics-assisted confinement (MAC) in picoliter droplets. We show that these homogeneous MAC-synthesized nanocomplexes exhibit narrower size distribution, lower cytotoxicity, and higher transfection efficiency compared to their bulk-synthesized counterparts. MAC represents a physical approach to control the energetic self-assembly of polyelectrolytes, thereby complementing the chemical innovations in nanocarrier design to optimize nucleic acid and peptide delivery.

CLINICAL TRIALS AND TRANSLATIONAL MEDICINE COMMENTARY: Drug Delivery Trends in Clinical Trials and Translational Medicine: Challenges and Opportunities in the Delivery of Nucleic Acid-Based Therapeutics

The ability to deliver nucleic acids (e.g., plasmid DNA, antisense oligonucleotides, siRNA) offers the potential to develop potent vaccines and novel therapeutics. However, nucleic acid-based therapeutics are still in their early stages as a new category of biologics. The efficacy of nucleic acids requires that these molecules be delivered to the interior of the target cell, which greatly complicates delivery strategies and compromises efficiency. Due to the safety concerns of viral vectors, synthetic vectors such as liposomes and polymers are preferred for the delivery of nucleic acid-based therapeutics. Yet, delivery efficiencies of synthetic vectors in the clinic are still too low to obtain therapeutic levels of gene expression. In this review, we focus on some key issues in the field of nucleic acid delivery such as PEGylation, encapsulation and targeted delivery and provide some perspectives for consideration in the development of improved synthetic vectors.

Design and development of polymeric nanoparticles for targeted delivery of nucleic acid-based therapeutics to tumor sites

Nucleic acids are widely used as potent therapeutics in cancer research. They can either promote gene expression by bringing a gene either not expressed or under-expressed into tumor cells (cDNA), or alternatively silence expression of genes such as oncogenes (RNAi mediators). However, before they can be efficiently translated to the clinic, this technology requires some optimization: nucleic acids and their vehicles need for instance to be protected from rapid elimination from the bloodstream (opsonization, clearance, and nuclease-mediated degradation) and the specificity of tumor addressing has to be validated. Hence a polymeric nanoparticular carrier encapsulating nucleic acids, either plasmid DNA or siRNA, was developed. Nanoparticles are composed of (1) PLGA, a well tolerated and biodegradable polymer, (2) PEG groups to avoid opsonization, (3) PEI moieties to complex nucleic acids and to enhance cytosolic delivery and (4) RGD sequence for active tumor targeting. Nanoparticles were formulated by double emulsion or water-in-oil-in-water method. Physical properties of such nanoparticles were assessed by dynamic light scattering (size and polydispersity index) and laser doppler electrophoresis (zeta potential). The efficiency of nucleic acid encapsulation into the carrier was determined by the Picogreen assay. Cytotoxicity and transfection capacity were assessed in an in vitro model of B16F10 melanoma cells. To date, various designs of nanoparticles were successfully formulated with appropriate size, surface charge and encapsulation efficiency. The PLGA nanoparticles did not show cytotoxic effects on cells and, although less efficient than PEI alone, allowed DNA delivery into tumor cells.

Nano-vectors for the ocular delivery of nucleic acid-based therapeutics

Nucleic acid-based therapeutics have gained a lot of interest for the treatment of diverse ophthalmic pathologies. The first to enter in clinic has been an oligonucleotide, Vitravene® for the treatment of cytomegalovirus infection. More recently, research on aptamers for the treatment of age related macular degeneration has led to the development of Macugen®. Despite intense potential, effective ocular delivery of nucleic acids is a major challenge since therapeutic targets for nucleic acid-based drugs are mainly located in the posterior eye segment, requiring repeated invasive administration. Of late, nanotechnology-based nano-vectors have been developed in order to overcome the drawbacks of viral and other non-viral vectors. The diversity of nano-vectors allows for ease of use, flexibility in application, low-cost of production, higher transfection efficiency and enhanced genomic safety. Using nano-vector strategies, nucleic acids can be delivered either encapsulated or complexed with cationic lipids, polymers or peptides forming sustained release systems, which can be tailored according to the ocular tissue being targeted. The present review focuses on developments and advances in various nano-vectors for the ocular delivery of nucleic acid-based therapeutics, the barriers that such delivery systems face and methods to overcome them.

Targeted Delivery of Nucleic Acid-Based Therapeutics to the Pulmonary Circulation

Targeted delivery of functional nucleic acids (genes and oligonucleotides) to pulmonary endothelium may become a novel therapy for the treatment of various types of lung diseases. It may also provide a new research tool to study the functions and regulation of novel genes in pulmonary endothelium. Its success is largely dependent on the development of a vehicle that is capable of efficient pulmonary delivery with minimal toxicity. This review summarizes the recent progress that has been made in our laboratory along these research directions. Factors that affect pulmonary nucleic acids delivery are also discussed.

Marrow-Derived Stromal Cells as Gene Transfer Vehicles in a Murine Model of Hemophilia A.

Marrow-derived stromal cells (MSCs) are considered a promising cellular vehicle for the delivery of nucleic acid-based therapeutics. In preclinical studies using a murine model of hemophilia A, we studied the effectiveness of ex vivo retrovirally-modified MSCs at synthesizing therapeutic amounts of factor VIII (fVIII). Previously, we observed robust anti-fVIII immune responses following intraperitoneal or intravenous delivery of MSCs genetically-modified to express a B-domain-deleted high expression porcine fVIII (HEP-fVIII) transgene (1). The presence of anti-HEP-fVIII antibodies masked any potential HEP-fVIII in circulation and thus, it was not determined whether or not the MSCs persisted in vivo. Furthermore, transplantation of HEP-fVIII-expressing MSCs into wild-type C57BL/6 mice induced an anti-fVIII immune response that decreased the endogenous murine fVIII levels to <10% of normal between wks 2 and 3 post-transplant. Analysis of plasma samples from these mice demonstrated the presence of anti-fVIII immunoglobulin populations that displayed cross-reactivity with human and murine fVIII in an ELISA and inhibited human and murine fVIII activity in a modified Bethesda assay. Therefore, pre-existing tolerance does not prevent the generation of antibodies that bind and inhibit murine fVIII. To further study the expression of HEP-fVIII from MSCs in immuno-suppressed animals, hemophilia A mice were pretreated with 5.5 Gy total body irradiation (TBI) prior to receiving HEP-fVIII-expressing MSCs. Pre-transplant TBI facilitated the detection of circulating fVIII activity for up to 6 wks, compared to 5 days in non-irradiated mice with an observed peak fVIII activity of 20 units/ml, which is 20-fold higher than the normal human level. However approximately 6 wks post-transplant, these mice developed large abdominal tumors that displayed histology similar to non-malignant fibrosarcoma. PCR analysis confirmed the presence of the HEP-fVIII transgene in the tumor cells at ~38 copies per diploid genome equivalent. In contrast shortly after viral transduction, the MSCs were shown to contain ~2 transgene copies per diploid genome equivalent, which increased to 5 and subsequently to 19 during ~1yr of expansion in culture. These results suggest that time-dependent genome amplification occurred during ex vivo culture and possibly in vivo post-transplantation. Cultured tumor cell explants secreted high levels of fVIII at a rate of 12 units/106 cells/24 hr in serum-free medium. Together these data indicate that the genetically-modified MSCs comprised a significant proportion of the tumor mass. The observation of tumor formation in these mice is consistent with previous reports describing a transformed phenotype in human and murine MSCs that had been cultured extensively ex vivo (2–4). The current findings demonstrate that, although murine MSCs are amenable to ex vivo manipulations and are capable of efficient fVIII biosynthesis, the immunogenicity of MSC-produced fVIII and the potential for oncogenic transformation raise important concerns regarding the use of MSCs as vehicles for gene therapy.

Novel Polyaminolipids Enhance the Cellular Uptake of Oligonucleotides

Two new polyaminolipids have been synthesized for the purpose of improving cellular uptake of oligonucleotides. The amphipathic compounds are conjugates of spermidine or spermine linked through a carbamate bond to cholesterol. The polyaminolipids are relatively nontoxic to mammalian cells. In tissue culture assays, using fluorescent-tagged or radiolabeled triple helix-forming oligonucleotides, spermine-cholesterol and spermidine-cholesterol significantly enhance cellular uptake of the oligomers in the presence of serum. Spermine-cholesterol is comparable with DOTMA/DOPE (a 1:1 (w/w) formulation of the cationic lipid N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) and the neutral lipid dioleylphosphatidylethanolamine (DOPE)) in increasing cellular uptake of oligonucleotides, while spermidine-cholesterol is more efficient. The internalized oligonucleotides are routed to the nucleus as early as 20 min after treatment, suggesting that the polyaminolipids increase the permeability of cellular membranes to oligonucleotides. At later times, much of the incoming oligonucleotides are sequestered within punctate cytoplasmic granules, presumably compartments of endosomal origin. Coadministration with polyaminolipids markedly improves the cellular stability of the oligonucleotides; more than 80% of the material can be recovered intact up to 24 h after addition to cells. In the absence of the polyaminolipids, nearly all of the material is degraded within 6 h. These data suggest that the new polyaminolipids may be useful for the delivery of nucleic acid-based therapeutics into cells.