Recent Advances in Nucleic Acid-Based Delivery

Review Journal of Biomedical Nanotechnology

Copyright © 2016 American Scientific Publishers All rights reserved Printed in the United States of America

Vol. 12, 841–862, 2016 www.aspbs.com/jbn

Recent Advances in Nucleic Acid-Based Delivery: From Bench to Clinical Trials in Genetic Diseases Cláudia Oliveira1 2 3 , António J. Ribeiro1 2 4 , Francisco Veiga4 5 , and Isabel Silveira1 2 3 ∗ 1

Group Genetics of Cognitive Dysfunction, i3s – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135, Porto, Portugal 2 IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180, Porto, Portugal 3 ICBAS – Universidade do Porto, 4050-313, Porto, Portugal 4 Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal 5 CNC – Center for Neuroscience and Cell Biology, Coimbra, 3004-504, Coimbra, Portugal

Delivery of nucleic acids is the most promising therapy for many diseases that remain untreatable. Therefore, many research efforts have been put on finding a safe and efficient delivery system able to provide a sustained response. Viral vectors have proved to be the most efficient for delivery of nucleic acids and, thus, stand as the foremost vector used in current clinical trials. However, safety issues arise as a main concern and mitigate their use, impelling the improvement of non-viral alternatives. This review focuses on the recent advances in pre-clinical development of non-viral polyplexes and lipoplexes for nucleic acid-based delivery, in contrast with vectors being used in present clinical trials. Delivered by Ingenta to: SUNY Upstate University cystic fibrosis, hemophilia, Nucleic acid vectors for neurodegenerative ataxias, Parkinson’s disease, Medical retinitis pigmentosa, IP: 5.8.47.242 On: Fri, 13 May 2016 20:50:34 pancreatic and lung cancer, and rheumatoid arthritis are discussed to illustrate current state of pre-clinical and clinical Copyright: American Scientific Publishers studies. Thereby, denoting the prospects for treatment of genetic diseases and elucidating the trend in non-viral vector development and improvement which is expected to significantly increase disease rescue exceeding the modest clinical successes observed so far.

KEYWORDS: Clinical Trials, Non-Viral Vectors, Nucleic Acid, Multivalency, Transfection Efficiency.

CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Non-Viral Nucleic Acid-Based Delivery . . . . . . . Non-Viral Vectors in Nucleic Acid-Based Therapy Delivery in Neurodegenerative Ataxia . . . . . . Brain Delivery in Parkinson’s Disease . . . . . . Retinal Delivery for Gene Replacement . . . . . Gene Replacement in Lung for Cystic Fibrosis Treatment . . . . . . . . . . . . . . . . . . . Gene Replacement in the Liver for Hemophilia Delivery in Pancreatic Cancer . . . . . . . . . . . Gene Delivery in Lung Cancer . . . . . . . . . . . Gene Silencing in Rheumatoid Arthritis . . . . . Unexpected Toxicity and Adverse Events in Gene Therapy Clinical Trials . . . . . . . . . . . . . . . . . . Challenges for Successful Clinical Application of Non-Viral Vectors . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . ∗

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Author to whom correspondence should be addressed. Email: [email protected] Received: 13 September 2013 Revised/Accepted: 8 January 2015

J. Biomed. Nanotechnol. 2016, Vol. 12, No. 5

It has now been over 20 years since the first clinical trial for gene therapy took place ameliorating the symptoms of the genetic disease adenosine deaminase deficiency severe combined immunodeficiency.1 There was hope that replacement of certain genes would then be applied to other diseases, which remained untreatable.2 Although the reasoning was correct, the actual work proved not to be that simple and, in the face of some severe complications and casualties, the ambient around gene therapy turned very apprehensive.3 Nucleic acid-based delivery bolstered when the mechanism of RNA interference (RNAi) in mammalians was described by Fire and collaborators, in 1998.4 The findings that RNAi can be mediated by double strand RNA (dsRNA) that are processed to molecules of 21–23 nucleotides, named small interfering RNAs (siRNAs), opened the door for a number of possibilities for silencing mutant genes known to cause disease.5 Since then, researchers have been trying to develop efficient strategies to deliver either siRNA

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Recent Advances in Nucleic Acid-Based Delivery: From Bench to Clinical Trials in Genetic Diseases

alone or short hairpin RNA structures (shRNA) which, after processing, are able to provide siRNA and silence a target mRNA, by inducing its degradation.6 These findings fueled research for specific application of this silencing mechanism and Xia and colleagues, in 2004, first demonstrated the therapeutic efficiency of RNAi in vivo for improvement of symptoms in mice with the neurodegenerative spinocerebellar ataxia type 1 (SCA1).7 Thereafter, the activity of RNAi resulting from siRNA therapy for

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the treatment of solid tumor cancers in humans was described and is being evaluated in an ongoing clinical trial.8 The premise that any defected gene can be targeted by designed synthetic siRNA or plasmid DNA (pDNA) which will be able to restore healthy functions, has driven many scientists to research in the field6 and allowed an upward development of therapeutic strategies. Even though problems have arisen during clinical trials, the largest obstacle has been delivery of nucleic acids.9–11

Cláudia Oliveira graduated in Pharmaceutical Sciences from the University of Coimbra, Portugal, in 2013. Currently she is a Ph.D. candidate in Molecular and Cell Biology from the Institute of Biomedical Sciences Abel Salazar, University of Porto, under the supervision and co-supervision of Professors Isabel Silveira and António Ribeiro. Her research work focuses on the development of non-viral vectors for treatment of neurodegenerative diseases.

António J. Ribeiro received his degree in Pharmaceutical Sciences in 1991 from the University of Lisbon, Portugal. He then pursued his studies in Pharmacokinetics and Pharmaceutical Technology at the University Paris XI in France. In 1997 he obtained his Ph.D. degree from University Paris V in Pharmaceutical Technology specializing Delivered by Ingenta to: under SUNYthe Upstate Medical in microencapsulation supervision andUniversity co-supervision of Professors J. C. IP: 5.8.47.242 On: Fri, 13 May 2016 20:50:34 Chaumeil and Ronald J Neufeld. His research work is multidisciplinary yet mainly Copyright: American Scientific Publishers focused on pharmaceutical development of drug delivery systems for proteins and peptide drugs through micro- and nanoencapsulation.

Francisco Veiga received his degree in Pharmaceutical Sciences in 1986 from the University of Coimbra, Portugal. He then pursued his studies in Pharmaceutical Technology at the University of Coimbra and University of Nancy, France. In 1996 he obtained his Ph.D. degree from University of Coimbra in Pharmacy specializing in Pharmaceutical Technology under the supervision and co-supervision of Professors Adriano Sousa and Philippe Maincent. He has published more than 120 peer-reviewed articles and several book chapters and patents. His publications have examined drugs enhancing bioavailability by using cyclodextrins and biopolymers.

Isabel Silveira graduated in Pharmaceutical Sciences from the University of Porto, Portugal, in 1990. She then pursued her studies in Molecular and Cellular Biology at the University Paris V in France. In 1997 she obtained her Ph.D. degree in human genetics specializing in disease gene mapping under the supervision and co-supervision of Professors Jorge Sequeiros, Guy Rouleau and Arnold Munnich. Her current work focuses on finding genes implicated in neurodegenerative diseases towards development of genetic diagnosis and treatment for these pathologies.

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Ideally, for a vector to allow an efficient transfection of problem.20 Recent advances in nanocarriers chemistry that cells it must be able to protect nucleic acids from degrahave yielded promising non-viral delivery systems have dation by nucleases in intercellular matrices and permit been reviewed by Yin and collaborators.21 12 targeting of the adequate cells and tissues, together with A number of PP and LP have been developed that offer great hope in overcoming the problems of their viral counthe internalization of RNA through the plasma membrane terparts concerning safety issues. In this review we focus into the cytoplasm and DNA further across the nuclear on non-viral polymeric and lipidic vectors for nucleic acids membrane into the nucleus of the cell.13 Furthermore, the delivery contrasting the strategies in various stages of clincarrier must present a good safety profile and not elicit ical evaluation for therapy of genetic diseases (Fig. 1); thus cytotoxicity. evidencing a major bottleneck in non-viral vector developThe first aspect to be taken into consideration for the ment which is clinical translatability. preparation of a new vector for nucleic acid based-delivery is the disease mechanism. For loss of function in autosomal recessively transmitted diseases, a gene replacement NON-VIRAL NUCLEIC ACID-BASED therapy has to be carried out, whereas for gain of funcDELIVERY tion pathologies with autosomal dominant pattern of inherNon-viral approaches for therapy of genetic diseases itance specific silencing of the mutant gene is the method can be divided into three groups: naked DNA, physical of choice. pDNA is used in gene replacement and, in the approaches, such as the hydrodynamics methods, gene latter, siRNA or similar structures, such as shRNA and gun and electroporation, and chemical methods, which 2 6 microRNA. mainly involve vectors that have cationic components in Furthermore, to guarantee that the genetic material is their composition, such as cationic liposomes and cationic able to reach target cells and organs, a suitable vector polymers.22 23 must be selected. Viruses excel in this task since evoNaked or pDNA exhibits low cellular uptake and rapid lution itself allowed for their capability of transfecting clearance. As a consequence, naked DNA injection is used cells very efficiently. Hence, many attempts of genetic when the low gene transfer efficiency can be relayed by correction made use of this type of carriers, most freimmune system activation and/or local bystander effects.24 14 quently adeno-associated virus, lentivirus and retrovirus. Regarding the physical methods, their potential to be Although some modifications were made in order to Delivered by Ingenta to: SUNY Upstate Medical Universityof gene therapy has already used in clinical applications extinguish their ability to induce IP: disease, this has 5.8.47.242 On:not Fri, 13been Maydemonstrated 2016 20:50:34 as shown by the approval of five cliniCopyright: been totally accomplished.15 Consequently, viral American vectors Scientific Publishers cal trials involving gene gun protocol.25 Nevertheless, furare associated with immunogenicity problems, as well as ther optimization of these nucleic acid delivery protocols insertional mutagenesis, and do not fully comply with will be needed in order to increase their efficiency without safety requirements. Additionally, there is a limitation in inducing a severe toxicity.22 23 size of the nucleic acids to be carried.16 Thus, reappraisal On the other hand, polymers and liposomes represent of these vectors was necessary, which led to enhanced the most extensively investigated and commonly used nondevelopment of non-viral vectors. viral nucleic acid-based delivery methods. These make use Designed to resemble viruses through encapsulation of of cationic polymers and lipids to incorporate DNA or nucleic acids with polymeric or lipidic material, polyplexes (PP) and lipoplexes (LP) respectively, are much simpler and cheaper to produce, allowing for easy scale transposition.17 These non-viral vectors present a greatly improved safety profile, compared to viral vectors, due to their low immunogenicity.18 However, the extent to which cells are transfected is quite lower16 and expression of the foreign gene or siRNA is transient,14 which stand as the greatest barriers to their use. When formulating a non-viral vector, it is also imperative to adequate the chosen nucleic acid to the appropriate vector. Indeed, although both molecules are able to complex with lipids or polymers, they do so in a differFigure 1. Innovative and rational formulations of non-viral ent manner, dependent on size, structure and chemistry of vectors carrying nucleic acids, particularly lipo- and polyplexes, have been described for the treatment of several the nucleic acid. Thus, pDNA condenses excellently with genetic diseases, overtaking their viral counterparts. Improved polymers due to its larger size, whilst siRNA establishes safety profiles and efficiencies of transfection have changed less electrostatic interactions, which results in a weaker the paradigm of pre-clinical studies and are impelling their conjugation.19 However, some modifications on the carrier progression into clinical trials, shedding light on gene therapy or siRNA itself have been described to try to surpass this future applications. J. Biomed. Nanotechnol. 12, 841–862, 2016

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RNA, so they are called PP, in the first case, and LP, in nucleic-acid delivery, schematically depicted in Figure 2, the latter.16 the most important are: (1) biomaterial binding/complexation/condensation of The assemblies of two oppositely charged macronucleic acid,40 41 molecules as are polynucleotides and cationic poly(2) cellular uptake/endocytosis,42 43 mers can lead to the formation of compacted particles (3) endosomal escape,44 through the establishment of attractive coulombic forces, (4) release of cargo/biomaterial unbinding,45 hydrogen bonds, hydrophobic, van der Waals and other 26 (5) biomaterial degradation,46 and in the case of DNA, interactions. Several polymers are described as being (6) nuclear import.47 suitable for PP formulation such as polyethylenimine 27 28 29 Firstly, establishment of a stable complex between the (PEI), polylysine (PLL), chitosan and dendrimers, nucleic acid and the delivery system is imperative. Not which are branching molecules that are slightly positively only is this fundamental to prevent enzymatic degradation charged.30 Whilst very useful for the possibility of surby nucleases but also to decrease the amount of interacface modification, the efficiency of these non-viral vections with serum proteins.19 Hence, behavioral studies of tors depends on their ability to condense the nucleic acid, 26 the carriers in whole blood are necessary.48 while allowing it to dissociate after internalization. It has recently been reported that polyanion/DNA/polycation ternary PP markedly improve DNA transfection activity in comparison with the original DNA/polycation binary PP.31 The original pDNA/polycation binary PP became clustered by strong adsorption of large amounts of serum proteins, leading to a sharp reduction in cellular uptake and intracellular accumulation, and thus low gene transfer efficiency. These results provide a basis for the development of polyanion/DNA/polycation ternary PP31 32 as well as DNA nanoencapsulation by alginate33 for PP stabilization against clustering by serum proteins. Lipidic systems includeDelivered liposomes, micellesto:and by Ingenta SUNY Upstate Medical University emulsions11 and are typically composed of at least IP: 5.8.47.242 On:two Fri, 13 May 2016 20:50:34 Copyright: elements: a cationic lipid and a neutral lipid,American which Scientific Publishers allows for stabilization of the former in a lipid bilayer and increased transfection efficiency.34 35 Most frequently, 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) and cholesterol are used as neutral lipids whereas N -[1-(2,3-dioleyloxy)propyl]-N ,N ,N -trimethylammonium chloride (DOTMA), 2,3-dioleyloxyN -[2(sperminecarboxamido)ethyl]-N ,N -dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dioleoyl3-trimethylammoniumpropane (DOTAP) and dioctadecylamido-glycylspermine (DOGS) are examples of commercially available cationic lipids.18 Liposomes Figure 2. Non-viral delivery of nucleic acids is essentially of distearoylglycerophosphatidyl choline (DSPC), dilihindered by a few critical steps that lead to ineffective noleyldimethylaminopropane (DLinDMA), methoxytransfection of cells. Establishment of a stable interaction polyethylene glycol carbamoyl-dimyristyloxy-propylamine between biomaterials and nucleic acids is fundamental for (PEG-c-DMA), and cholesterol were successfully the success of the formulation and diminishing unspecific employed in systemic delivery of siRNA.36 The formulainteractions following carrier administration (1). This is also relevant regarding carrier size and charge since interaction tion of both LP and PP for nucleic acid delivery have been 26 with the cell membrane is critical for the internalization prothoroughly reviewed by Tros de Ilarduya and colleagues. cess, which primarily occurs through endocytic mechanisms There are two main problems associated with devel(2). Following cellular uptake, efficient release of the carriopment of non-viral vectors: poor transfection efficiency ers from endosomes has to occur in order to avoid degradaand low transgene expression.37 38 Extensive efforts have tion in lysosomes (3). However, successful transfection may be impaired by inappropriate biomaterial unbinding, nuclease been made to improve non-viral nucleic-acids delivery effidegradation of nucleic acids in the cytoplasm or their inabilciency through rational and semi-rational design of vectors ity to reach the target organelle (4). Indeed, siRNA needs to by considering the specific mechanistic steps of delivtrigger a mechanism of mRNA degradation in the cytoplasm ery that are capable of overcoming the various transfec(5) whereas pDNA requires nuclear translocation in order to tion barriers.39 Among the several key steps in non-viral initiate the subsequent steps for DNA expression (6). 844


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It is generally accepted that electrostatic interactions time to either escape the endosome or mediate endosodrive the interaction between cationic non-viral vectors mal escape.63 Other strategies that have been utilized to 49 and the net negatively charged cell surface provided by promote endosomal escape include functionalizing polyproteoglycans carrying various glycosaminoglycan chains, mers with endosomolytic peptides,64 which make use of 50 including heparan, dermatan, and chondroitin sulfates. pH sensitive conformational changes that promote endoParticularly heparan sulfate proteoglycans (HSPGs), to somal escape. which transmembrane proteins like syndecans belong as However, non-viral vectors employing the proton prominent family members, are considered as major bindsponge mechanism have some drawbacks such as a high ing sites for cationic delivery vectors.51 52 cellular toxicity, as they are typically membrane-lytic.65 Recent evidence, obtained by confocal microscopy and Additionally, delivery vectors may be released into the live cell imaging, in conjunction with scanning electron cytoplasm early in the transfection process, where they microscopy, demonstrates that after initial binding of LP face degradation by nucleases.66 Furthermore, after leavor PP, rapid clustering of syndecans takes place, stabiing endocytic vesicles, the nucleic acids have little or no lizing the binding of the nanovectors, which is followed active transport to the target organelle; studies have shown by their surfing along or retraction by filopodia, i.e., thin, that DNA intracellularly microinjected into the cytoplasm actin-rich cellular extensions of several microns in length. undergoes little to no diffusion.67 68 Interference with actin polymerization or inhibition of the Following nuclear import, important downstream steps motor protein myosin II, localized at the base of filopodia, for DNA expression include promoter binding to the revealed53 that the binding of the nanovectors to and subDNA, transcription to mRNA, and translation into prosequent clustering of syndecans initiates actin retrograde tein. Although these mechanistic steps have been identiflow, which moves the syndecan-bound nanovectors to the fied, they are still not well understood and further research cell body, analogously as reported for viral particles.54 is necessary to elucidate how biomaterial structure preAmongst a variety of pathways, endocytosis is curcisely affects each step.69 rently considered the primary pathway by which non-viral Formulation of nucleic acids must reflect the place nucleic acids delivery vectors are internalized,42 although of their action, in order to account for the difficulties some reports have suggested that non-viral nucleic acids that must be overcome for the target to be reached. 55 delivery has a non-endocytic Delivered mechanism.by There areto: mulinstances, the University biological interactions elicited upon Ingenta SUNYFor Upstate Medical tiple endocytic pathways including IP: phagocytosis well 5.8.47.242asOn: Fri, 13systemic May 2016 20:50:34 impact greatly on the safety of administration 57 Copyright: 56 American Publishers as clathrin- and caveolae-dependent endocytosis, but Scientific therapeutics as well as their efficiency.70 Further probrecent studies have failed to demonstrate the dominance lems associated with the design of non-viral vectors such of any particular internalization processes that lead to as their physical and chemical influence on transfectransfection.58 tion efficiency are excellently reviewed by Wang and In the internalization of non-viral vectors, it is imporcollaborators.71 tant to take into account release from endosomal comTo overcome the aforementioned obstacles, many innopartments into the cytoplasm, which may further depend vative strategies have been developed, endowing vectors on the nature of the vector.59 LP are suggested to escape with targeted delivery,72 less toxicity73 74 and improved from the endosome through a mechanism that involves transfectivity.63 Particular measures include the coupling of lipid mixing, which relies on the ability of cationic lipids, targeting peptides, integrin, transferrin and folate as well alone or in the presence of helper lipids, to form nonas glycosylation and conjugation of antibodies or growth bilayer structures. This transformation appears to require factors.16 Furthermore, surface modification with polymer a LP-induced flip-flop mechanism within the endosomal coatings,10 hemifluorination of polycationic lipid, which membrane which leads to the establishment of neutral confers the LP higher stability,75 and the use of lipids able ion pairs with cationic lipids that induce local destabito respond to stimuli (functional lipids)18 have also been lization, allowing nucleic acid dissociation and release.60 reported, amongst others. Conversely, it has been shown that polymer/DNA particles can escape the endosome into the cytoplasm through the NON-VIRAL VECTORS IN NUCLEIC “proton sponge effect.”61 Inside of the endosome, the pH ACID-BASED THERAPY drops from 7.4 to around 5.0, where a polymer’s secondary 62 Improved non-viral vectors for delivery in a wide range and tertiary amine groups can buffer the acidification. of genetic diseases affecting specific organs have been An influx of ions into the endosome can lead to osmotic described. Table I summarizes some of the most recent swelling and eventual bursting to release the PP into the non-viral vectors specifically designed for delivery of cytoplasm.44 The buffer capacity of titratable amine groups pDNA or siRNA to cells and organs affected in represencan effectively facilitate endosomal rupture, inducing effitative genetic conditions that reflect recent progresses in cient gene expression.62 Studies have shown that polymers the field. with secondary or tertiary amines are able to provide more J. Biomed. Nanotechnol. 12, 841–862, 2016

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Recent Advances in Nucleic Acid-Based Delivery: From Bench to Clinical Trials in Genetic Diseases Table I.

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Recent non-viral vectors for nucleic acid based-delivery.

Clinical setting

Nucleic acid

Gene

Vector

Delivery route

siRNA

ATXN1

PEG-PP-TAT

Neuro2a cell incubation∗∗

83 84

pDNA

GDNF

NTS1-PLL PP

Retinitis pigmentosa88 89

pDNA pDNA pDNA

GDNF Rds EGFP∗

Lactoferrin PP CK30PEG PP PBAE PP

Injection to the substantia nigra and ipsilateral striatum/rat Intra-peritoneal/rat Subretinal/mice Subretinal/mice

Cystic fibrosis99–101

pDNA pDNA pDNA

EP Firefly luciferase protein∗ EGFP∗ lacZ∗

PEI PP CL LP CL NPh LP

Hemophilia104 105

pDNA siRNA pDNA siRNA siRNA

F8 AT EGFP HER-2 VEGF

Chitosan PP GalNAc EGFR-Gelatin PP TfR-LP Calcium phosphate PP

Oral/mice Subcutaneous/mice PANC-1 cell incubation∗∗ Intravenous/mice Intravenous/mice

Lung cancer116–119

pDNA siRNA siRNA siRNA

AT2-R MDM2; c-myc; VEGF PLK-1 PHB1

dTAT PP LCP PP ATCOL PP PEG-NPh CL PLGA PP

Intratracheal/mice Intravenous/mice Intravenous/mice A549 cell incubation∗∗

Rheumatoid arthritis126–128

siRNA siRNA siRNA

TNF- IL-1;IL-6;IL-18 COX-2

PLGA CL PP PLGA LP PEI PGLA PP

SCA80 Parkinson’s disease

Pancreatic cancer109–111

Pulmonary/murine Intravenous/mice Bronchial cell incubation∗∗ Intratracheal instillation/mice

Intra-articular/murine Intravenous/mice C28/I2 cell incubation∗∗

Notes: SCA—Spinocerebellar ataxia; pDNA—Plasmid DNA; siRNA—Small interfering RNA; ATXN1—Ataxin-1; GDNF—Glial cell-derived neurotrophic factor; Rds—Retinal degeneration slow; EGFP—Enhanced green fluorescent protein; ∗ —Reporter gene; EP—Erythropoietin; lacZ—E. coli lacZ gene encoding -galactosidase; F8—Factor 8; AT—Antithrombin; HER-2—Human epidermal growth factor receptor 2; VEGF—Vascular endothelial growth factor; AT2R—Angiotensin type 2 receptor; MDM2-c-myc-VEGF—Mouse double minute 2 homolog—proto-oncogene homologous to myelocytomatosis virus—vascular endothelial growth factor; PLK-1—Polo-like kinase 1; PHB1—Prohibitin 1; TNF-—Tumor necrosis factor-alpha; IL—Interleukin; COX-2—Ciclo-oxigenase-2; Delivered bytrans-activator Ingenta to:of SUNY Upstate University PEG—Polyethylene glycol; PP—Polyplex; TAT—HIV-1 transcription peptide;Medical NTS-1—Neurotensin receptor; PLL—Polylysine; CK30PEG— IP: 5.8.47.242 On: Fri, 13 May 2016 20:50:34CL—Cationic lipid; LP—Lipoplex; NPh—Neutral Polyethylene glycol-substituted 30-mer lysine peptides; PBAE—Poly(beta-amino ester); PEI—Polyethylenimine; phospholipid; GalNAc—Asialoglycoprotein receptor ligand derived from N-acetylgalactosamine; EGFR—Epidermal growth factor receptor; TfR—Transferrin Copyright: American Scientific Publishers receptor; dTAT—Dimerized HIV-1 trans-activator of transcription peptide; LCP—Lipid/calcium/phosphate; ATCOL—Atelocollagen; PLGA— Poly(lactic-co-glycolic acid); Neuro-2a—Mouse neuroblastoma cells; ∗∗ —Study only performed in vitro; PANC-1—Human pancreatic carcinoma, epithelial-like cell line; A549—Human lung adenocarcinoma epithelial cell line; C28/I2—Human chondrocyte cell line.

Delivery in Neurodegenerative Ataxia The spinocerebellar ataxias (SCAs) are a heterogeneous group of dominantly inherited brain diseases caused by varied types of mutations and pathogenesis.76 They are characterized by progressive gait, limbs and speech incoordination. In several of these pathologies, the affected can be genetically distinguished form the normal gene and targeted for silencing.77–79 Delivery to the brain is considerably more difficult to achieve than other organs as the blood brain barrier (BBB) tightly controls the passage of molecules. To facilitate crossing of the BBB, receptor targeting antibodies and cell-penetrating peptides have been used. In SCA1, silencing of the mutant allele using a viral vector improved motor symptoms and restored neuronal morphology in a mouse model.7 Recently, successful silencing of the ATXN1 gene, involved in SCA1, was obtained with a non-viral nanoparticle. In this study, a functional siRNA was delivered into mouse neuroblastoma cells by a PEGchitosan polymer conjugated to TAT-peptide, which promotes BBB transcytosis due to its strong cell adherence and ability to destabilize the lipid bilayer of the cell 846

membrane through a process independent of receptors and temperature.80 Brain Delivery in Parkinson’s Disease Parkinson’s disease involves degeneration of the dopaminergic neurons and culminates in the loss of dopamine in the striatum, although different genetic loci can be implicated in the mechanism of pathogenesis.81 Ultimately, patients start to experience symptoms of dyskinesia, resting tremor, rigidity and impaired postural reflexes.82 In an attempt to improve non-viral vectors for the treatment of Parkinson’s disease, Gonzalez-Barrios and colleagues have developed conjugates of neurotensin with PLL PP to carry the glial-derived neurotrophic factor (GDNF) gene into nigral dopamine neurons. The carrier uses the endocytosis of neurotensin with its high affinity receptor to shuttle the transgene into dopamine neurons. This study was the first to demonstrate the efficiency of these vectors, in a rat model, as evidenced by the presence of the GDNF protein in the striatum and substantia nigra of the rat brain. Improvement in motor function of rats with hemiparkinsonism was observed only with J. Biomed. Nanotechnol. 12, 841–862, 2016

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injection of the carrier with GDNF when levels of striatal dopamine increased.83 However, this carrier has limited use in humans because PLL was shown to induce apoptosis in cultured cells. Other brain-targeting non-viral vectors capable of crossing the BBB are lactoferrin (Lf)-modified nanoparticles. The large advantages of these non-viral vectors, their ability to cross the BBB and thus avoid invasive procedures such as intracerebral injection, was compromised with multi-dose administration since there is a short-term expression associated with non-viral vectors and effective concentrations have to be maintained. Expression of GDNF resulted in efficient and safe brain-targeted delivery. Moreover, behavioral studies demonstrated reversal of motor impairment along with decreased time of inactive sitting.84 The group has also reported angiopep-conjugated dendrigraft PLL nanoparticles suggested to be more capable of BBB transcytosis and parenchymal accumulation due to the ability of the ligand to specifically bind to low density lipoprotein receptor-related protein which is overexpressed on the BBB.85

degrade in water. Many of these polymer formulations showed transfection efficacy that was significantly superior to Lipofectamine 2000 and FuGeneHD, two of the lead commercially available alternatives for non-viral gene delivery, with comparable cellular viability.89

Gene Replacement in Lung for Cystic Fibrosis Treatment Cystic fibrosis is the most common autosomal recessive disease among the Caucasian population and is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.90 91 The dysfunctional CFTR protein leads to a decrease in airway surface liquid since there is a perturbation in ion transport92 which accrues for defective mucociliary clearance. Consequently, patients have extremely viscous mucus which leads to chronic and repetitive bacterial infections.93 Given the ease of access, the pulmonary tract has been the only administration route used so far although, evidently, the high viscoelasticity of the sputum stands as the greatest barrier for delivery.92 Most recent research focuses especially on the improvement of non-viral vectors since, Retinal Delivery for Gene Replacement for instance, viral vectors have higher risks in multiple administrations.94 Inefficiency of delivery is also a great Hereditary retinal blindness is a group of genetic diseases problem and some non-viral formulation strategies report caused by mutations in genes expressed in photoreceptors the use of surface modifications, as is coating with PEG, or the retinal pigment epithelium.86 87 Despite the phenoorder toMedical enhanceUniversity mucus penetration.95 In addition, Suk typic diversity, retinitis pigmentosa consists in progressive Delivered by Ingenta to: SUNYinUpstate degeneration of the retina culminating the apoptosis colleagues have improved particle uptake by combiIP: in 5.8.47.242 On: of Fri, 13and May 2016 20:50:34 Publishers national use of PEG and the mucolytic N-acetyl-cysteine photoreceptors that leads to blindness.Copyright: American Scientific establishing a decreased immobilization of particles in the Although work on viral vectors has been more extensive in these pathologies, there are some strategies being mucus.96 Notwithstanding, plasmid optimization has also developed to replace them by non-viral vectors. Cai and been a great priority, as even the presence of specific DNA sequences known as CpG islands is enough to trigger an collaborators have shown that compacted DNA nanoparticles are effective in partially rescuing the structural and immune response. For that reason, CpG-free plasmids have functional damage caused by retinitis pigmentosa in rds+/− been developed with positive results.97 These data, clearly 88 evidence that plasmid optimization is crucial for a successmice model. In view of delivering pDNA encoding the ful delivery and recent research has provided very insightwild-type retinal degeneration slow (Rds) glycoprotein, ful improvements.98 nanoparticles compacted by polyethylene glycol (PEG)substituted 30-mer lysine peptides (CK30PEG) were Furthermore, other groups have worked on dry powdesigned. Accordingly, these carriers are capable of a high der aerosols of polyethylenimine (PEI)-based gene vectors transfectivity because of their reduced diameter of less with sugar as lyoprotectant.99 Lyophilized particles had a than 8 nm and thus, are a viable alternative to viral vecmean diameter of 50–100 nm and retained their biological tors. Subretinal injections of these particles provided an activity. It was also established that the content of sugar in the formulation was crucial for protein expression and effective transfection of both mitotic and post-mitotic retinal cells and protein expression was detected in both rods particle size.99 and cones. There was evident structural improvement in Cationic lipophosphoramidates with two phytanyl virtually all photoreceptors observed as well as a thickchains for the in vivo transfection of pDNA have also been described.100 Lipid particles had mean diameters of ening of the outer nuclear layer. These results accounted for a significant decrease in photoreceptor death with pro200–250 nm and a hexagonal phase conferred higher fusotein expression up to four months post-treatment, without genic properties. A luciferase-expressing plasmid was used obvious adverse events.88 as a reporter gene for the transfectivity of these carriers, which targeted pneumocytes, as assessed through bioluPoly(beta-amino) ester-based nanoparticles are also minescence. Little or no toxicity was observed in vitro promising for delivery of pDNA to retinal pigment epithewhereas, in vivo, mild elevation of transaminases was lial cells. These nanoparticles are small in size (180 nm), reported albeit transient.100 have a positive zeta potential (+26 mV), and easily J. Biomed. Nanotechnol. 12, 841–862, 2016

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Optimization of delivery formulations may also be achieved with targeting peptides. In view of that, receptortargeted nanocomplexes comprising epithelial-targeting peptides, cationic liposomes and pDNA were developed.101 The similarity between part of the peptide and the targeting sequence of some intracellular respiratory pathogens allows for specificity, whereas another moiety is important for DNA packaging and promoting the interaction with nuclear importins. Shearing forces during nebulization did not compromise the biologic activity of the pDNA which was confirmed from the expression of the enhanced green fluorescence protein (EGFP) reporter gene in vitro.101

Tumor gene therapy consists in altering the neoplastic phenotype of cancerous cells or to provide additional protective effects, through delivery of genetic constructs.109 In early development studies an increasing number of nonviral vectors have been optimized to target these tumors. Type B gelatin-based engineered PP were developed for delivery of EGFP reporter pDNA.109 The surface of PP was modified with an epidermal growth factor receptor (EGFR)-targeting peptide. The EGFR-targeting peptide was grafted on the surface of PP using a PEG spacer to allow for longer systemic circulation time, passive targeted delivery by the enhanced permeability and retention effect and maximizing the flexibility on the binding of peptide and receptor. Pancreatic tumors are associated with Gene Replacement in the Liver for Hemophilia increased expression of EGFR thus peptide conjugation Hemophilia is a bleeding disorder characterized by a will allow for effective targeting. In this study, when transdeficit in either coagulation factor VIII (FVIII) or coagfected in EGFR-expressing Panc-1 cells, particles were ulation factor IX (FIX) of the coagulation cascade. Coninternalized up to 48% with relatively no toxicity.109 This sequently, patients often experience bleeding incidents, EGFR-targeting gelatin-PP carrier is a promising system depending on the severity of their disease.102 Concurfor nucleic acid delivery and transfection in human panrently, this genetic deficiency is intimately correlated with creatic cancer cells. co-morbidities, mainly chronic arthropathy.103 A nanosized immunoliposome-based delivery comDevelopment of non-viral vectors for treatment of plex (scL) carrying an anti-HER-2 siRNA molecule was hemophilia is seldom reported. However, the preparation developed as a potential selective inhibitor of tumor of chitosan nanoparticles was described for oral delivery growth.110 Inclusion of a pH-sensitive histidine-lysine pepof F8 transgene, incorporated in gelatin to favor feeding 104 tide in the complex allowed for increased potency, tumor to hemophilia A mice. Chitosan proved to induce the sensitization for chemotherapy and growth inhibition. The least toxicity. Improvement of this method by decorating to: SUNY Upstate Medical University nanoimmunoliposome complexes (scL) are composed of nanoparticles with PEG andDelivered inclusion by of Ingenta sugars may be IP: 5.8.47.242 On: Fri, 13 May 2016 20:50:34 required to prevent particle aggregation. Overall, protein siRNA encapsulated by a cationic liposome, the surface of Copyright: American Scientific Publishers expression levels of 1–4% were detected in the plasma which is inserted with an anti-transferrin receptor (TfR) which, despite modest, are sufficient to elicit bleeding single-chain antibody fragment to target the complex phenotype correction.104 A new non-viral vector known to both primary and metastatic disease. The small size as ALN-AT3 is composed of siRNA targeting antithrom(approximately 100 nm) and TfR-tumor-targeting nature bin (AT) conjugated to an asialoglycoprotein receptor ligof these particles resulted in enhanced cellular uptake by and derived from N -acetylgalactosamine (GalNAc) that human pancreatic cells.110 mediates binding and internalization by hepatocytes and is An alternative strategy for delivery of siRNA into being developed for the treatment of hemophilia and rare tumor cells makes use of calcium phosphate (Ca-P)bleeding disorders. Pre-clinical data showed that ALNbased nanoparticles.111 These nanocarriers were targeted AT3 demonstrated normalization of thrombin generation to subcutaneous pancreatic tumors by using anti-vascular and improvement of hemostasis in hemophilia models, endothelial growth factor (VEGF) siRNA, since this showing that ALN-AT3 can fully correct thrombin genermolecule is known to be over-expressed in many canceration in large animal models. Additionally, by administerous cells. Addition of a block copolymer of PEG and a ing highly exaggerated doses of ALN-AT3 to wild type charge-conversional polymer conferred enhanced colloidal and hemophilia animals, RNAi therapeutic revealed a wide stability and biocompatibility, brought upon by the PEG therapeutic index in the hemophilia setting.105 capsule, and endosome-disrupting functionality, due to the acidic pH-responsive anionic moiety of the polyanion actiDelivery in Pancreatic Cancer vated through cis-aconitic amide cleavage. Overall, tumor growth was significantly suppressed by direct RNAi effect Pancreatic cancer stands out among others because, with no observed severe or acute toxicity.111 The translaalthough infrequent, it is one of the top five most comtional capability of this delivery system was recently conmon causes of cancer mortality in most developed counfirmed using a spontaneous tumor model which resembles tries, due to its very poor prognosis.106 For the majority more closely the tumor microenvironment and cell popuof cancers, improved treatments and earlier diagnosis have lations. Indeed, in vivo silencing efficiency was approxireflected on better survival rates. However, for pancreatic mately 60% and no severe acute toxicity was induced as cancer these rates have not improved significantly over the last 25 years.107 108 Thus, there is a clinical demand for observed by indicators of liver and kidney function as well alternative and more efficient diagnosis and therapies. as expression of inflammatory cytokines.112 848


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Gene Delivery in Lung Cancer shell and a relatively neutral charge lipid-PEG surface layer has been reported to accomplish prohibitin 1 silencDespite a decrease in mortality due to reduction of tobacco ing in A549 lung cancer cells over 2 weeks, with gene consumption, lung and bronchus cancer is still the lead expression only recovering after 24 days. The protein cause of cancer-related death for both genders, in many encoded by this gene is involved in cell proliferation, apopgeographic regions.108 Lung cancer is a very heterogeneous disease; subsequently there are different molecular tosis and chemoresistance, among others, which evidences targets involved in disease progression. Although there has its potential for cancer treatment. Accordingly, xenograft been great advancement on lung cancer treatments, there is tumor proliferation in vivo was significantly arrested.119 still a significant lack of success, especially for non-small cell lung cancer, where there is often drug resistance.113 114 Gene Silencing in Rheumatoid Arthritis New and improved treatments are urgent and nucleic acidRheumatoid arthritis is an auto-immune disease charbased delivery may be an important strategy for tumor acterized by persistent synovitis, systemic inflammation suppression.115 and the presence of auto-antibodies.120 These progresAngiotensin II type 2 receptor (AT2R) is known to sive symptoms lead to permanent degeneration of joints inhibit cell proliferation and induce apoptosis in specific and are also correlated with an increased cardiovascutissues.116 Thus, a formulation of nanoparticles made from lar risk.121 Treatment for rheumatoid arthritis has signifithe dimerized TAT (dTAT) peptide from HIV-1, that reprecantly evolved through the past years, leading to delayed sents a protein transduction domain and a nuclear localizadisease onset and increased quality of life. Currently, tion sequence expected to increase transfection efficiency, disease-modifying anti-rheumatic drugs (DMARD) are was developed enclosing pDNA encoding AT2R. After the most significant option available, being methotrexate intratracheal administration to Lewis lung cancer bearmore frequently used.122 However, there is heterogeneous ing mice, it was possible to conclude that robust transresponse to these drugs and a great deal of adverse events gene expression was achieved with minimal toxicity, and associated,123 which evidence the need for further treatmaintained over a period of 14 days. Furthermore, results ment optimization. Nucleic acid gene therapy may be an suggest that dTAT-AT2R efficiently decreased tumor mulimportant alternative to DMARDs aiming for persistent 116 tiplicity and size. anti-inflammatory activity.124 The lung is recognized as being prone for metastaOne of Medical such approaches is the use of poly(DL-lactideby Ingenta SUNY Upstate University sis because of its oxygen Delivered rich environment andto: high co-glycolide) (PLGA) microspheres carrying siRNA IP: 5.8.47.242 On: Fri, 13 May 2016 20:50:34 vascularization. For systemic administration, were develCopyright: American Scientific Publishers against tumor necrosis factor (TNF- which is a welloped lipid/calcium/phosphate (LCP) nanoparticles coated established target in the treatment of rheumatoid arthriwith cationic lipids and grafted with PEG and anisamide, tis because of its prominent role among other cytokines. which target a receptor overexpressed in melanoma cells. A technology recently developed benefits from speThese particles encapsulate a combination of three differcific siRNA delivery within arthritic joints in order to ent siRNA sequences against MDM2, c-myc and VEGF. avoid systemic side effects and also a prolonged inhiMDM2 is an important regulator of p53; c-myc is a tranbition of TNF- which allows for improved results and scription factor involved in multiple functions, namely longer administration intervals.125 Correspondingly, PLGA proliferation and cell cycle control, genetic instability nanoparticles targeting TNF- have also been combined and sensitization to apoptotic stimuli; and VEGF is a with DOTAP, shown to increase transfection efficiency. growth factor that mediates tumor angiogenesis and metasAfter intra-articular injection in mice, magnetic resonance tasis. Intravenous injections of the LCP nanoparticles imaging showed a significant reduction of paw scores and to metastatic mice did not elicit immune responses and joint effusion but lack of amelioration of bone marrow allowed for efficient inhibition of tumor growth at low edema, which suggests the need for further studies on sysdoses.117 Conversely, lung cancer may also metastasize to temic intravenous administration.126 other sites such as the liver. In view of that, atelocollagen Similarly, a study was conducted comparing treatwas used as a carrier of siRNA to a liver metastatic mouse ment with siRNA against TNF- and siRNA cocktail model, targeting polo-like kinase 1 (PLK-1), an important co-targeting IL-1, IL-6 and IL-18, after systemic delivintervenient in cell division which is overexpressed in lung ery. It was possible to conclude that the best results were cancer tissues. Results suggest this formulation leads to achieved when the siRNA cocktail treatment was adminspecific inhibition of the growth of lung cancer cells and istrated reducing all features of the disease, including liver metastatic tumor growth in vivo, without inducing the inflammatory process, in mice with collagen induced innate immune responses.118 arthritis. Thus, cocktail combinatory treatment appears Furthermore, it is noteworthy the development of to be a relevant alternative to TNF- neutralization and siRNA-sustained release strategies for cancer treatment. reduction.127 A hybrid lipid-polymer nanoparticle composed of an aqueFurthermore, co-delivery of anti-COX2 siRNA and dexous siRNA core stabilized by a positively charged lipidamethasone has been studied as a potential enhanced like compound, a middle hydrophobic PLGA polymer J. Biomed. Nanotechnol. 12, 841–862, 2016

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treatment of rheumatoid arthritis. In view of that, PLGA nanoparticles loaded with dexamethasone were afterwards complexed with PEI/siRNA and delivered to a human chondrocyte line. Results suggested that the association provided the greatest reduction of inflammatory factors expression, in comparison with administration of antiCOX2 siRNA or dexamethasone alone. Cell viability was not decreased by combination of the drug with the nucleic acid which supports its use for further research.128 A more recent strategy employs a nanocomplex of polymerized siRNA targeting TNF- with thiolated glycol chitosan polymers for treatment of rheumatoid arthritis. Polymerization of siRNA improves charge–charge interactions with polymers due to the low charge density and short chain length of this nucleic acid and results from the 5 -end thiol modification of both sense and antisense strands generating poly-siRNAs with reducible disulfide linkages. In vivo studies demonstrated comparable efficiency to methotrexate without undesirable cytotoxicity and non-specific targeting.129

of the transgene proved to be permanent even though there is ongoing dopaminergic neuron degeneration, evidenced by decline of AADC-specific tracer levels. This treatment results in expression of AADC in the putaminal medium spiny neurons that do not degenerate unlike nigral neurons responsible for endogenous AADC expression, which poses as an important advantage. Notwithstanding, these results enhance the need for a controlled efficacy trial and higher vector dosages.132 Retinitis pigmentosa is a disease that, unlike the previous, has very limited clinical trial results. There are some therapies for the retina being assessed however they usually correlate to retinal degeneration in general. Cystic fibrosis clinical trials distinguish themselves amongst other gene-based therapies because many carriers used are already non-viral. In 1999, Alton and colleagues described a trial using a LP administered by nebulization with the intent of transferring the CFTR gene to the nasal epithelium of patients. Despite finding some mild respiratory symptoms, the carrier successfully accomplished reduction of inflammatory cells in the sputum, bacterial adherence and an improvement of approxUNEXPECTED TOXICITY AND ADVERSE imately 20% of the chloride abnormality. Contrariwise, EVENTS IN GENE THERAPY sodium hyper-absorption remained unaltered.133 The host CLINICAL TRIALS immune response may also be problematic: for tested LP Clinical approaches using nucleic acid-based therapy have (NCT00004806), pDNA which has been in common use is been hindered because a set of problems usually arise in generally rich in unmethylated CpG dinucleotides, which Delivered bytoIngenta SUNY are Upstate University the long way of pre-clinical development clinical to: trials. likely Medical to be recognized by humans as foreign and thus IP: 5.8.47.242 On: Fri, 13produce May 2016 20:50:34 Table II discriminates updated clinical trials data, namely an inflammatory response.134 This is the scientific Publishers the delivery system used, target geneCopyright: as well as American delivery Scientific basis reported for the ongoing clinical trial using a combiroute and phase of investigation. Taking into consideration nation of a pDNA expressing CFTR with a cationic lipothat most clinical trials here reported are still in phase 1, some for nebulized administration (NCT01621867) where the useful information about efficiency and safety is quite an improved plasmid with codon-optimized CFTR gene scarce, yet its availability will increase as clinical phases provides longer transgene expression in absence of inflamare overcome. matory effects.97 Clinical trials for Parkinson’s disease are proving to Prospects for hemophilia B treatment are encouraging be very insightful. The results obtained after each study with results from phase 1 clinical trial (NCT00979238) involving delivery of neurturin (NTRN) are thoroughly revealing that AAV vector expressing a codon-optimized described and have greatly contributed for technical human factor 9 (F9) transgene when administered by improvements. Initially, a phase 1 open-label trial demonperipheral vein delivery resulted in FIX expression suffistrated a significant improvement in 8 of 12 patients cient to improve the bleeding phenotype. Neither immune enrolled with absence of persistent adverse events. The responses to the transgene nor presence of neutralizing following studies, including a phase 2a trial, supported antibodies to FIX were registered, but the same did not the previous results; however primary outcomes were not verify for the T-cell reactivity to AAV8-capsid which was sufficient to conclude on treatment benefit. Nevertheless, dose and patient-dependent.135 Furthermore, ALN-AT3 has almost all endpoints favored NRTN over sham surgery, been reported with positive results from a phase 1 clinical establishing “proof of concept.” Phase 2b (NCT00985517) trial (NCT02035605). ALN-AT3 subcutaneously adminisis currently active.130 131 A study of convection enhanced tered allowed up to 57% knockdown of AT in hemophilia delivery of AAV2-GDNF to the putamen in subjects with subjects with duration of about 60 days after a single advanced Parkinson’s disease is in recruiting phase 1 dose. Results show ALN-AT3 was well tolerated in healthy (NCT01621581). volunteers and hemophilia patients.136 This therapeutic Additionally, a long-term follow up study on has also been granted orphan drug designation by the Parkinson’s disease (NCT00229736) of an AAV2 encodEuropean Medicines Agency.137 ing human aromatic L-amino acid decarboxylase (AAV2In pancreatic cancer, a study combining suicide gene hAADC) reported an acceptable safety profile for the therapy with chemotherapy used an adenovirus for intratumoral injection (NCT00415454). There is strong evidence treatment. As an improvement to other studies, expression 850



CFTR CFTR CFTR F9 F9 F9 F9 AT siRNA

Cystic fibrosis

p53 p53 TUSC2 TNFR:Fc

AdV AdV CL-NPh LP AAV2

Surface injection Endobronchial IV Intra-articular

Intratumoral Intratumoral Intratumoral IV

Nebulization Syringe instillation Intranasal IV IV Infusion Intra-hepatic Subcutaneous

Subretinal Subretinal Subretinal

Intraputaminal and intranigral CED to putamen Striatal Intrastriatal infusion

Delivery route Ceregene NINDS Oxford Biomedica Genzyme

Phase 2 Phase 1 Phase 1/2 Phase 1

Phase 2 Phase 1 Phase 1 Phase 1/2

Phase 1 Phase 1 Phase 1/2 Phase 1/2

Phase 2 Phase 1 Phase 1 Phase 1/2 Phase 1/2 Phase 1 Phase 1 Phase 1

Phase 1 Phase 1/2 Phase 1/2

Shenzhen SiBiono GeneTech Co. East. Coop. Oncol. Group M.D. Anderson Cancer Center Targeted Genetics Co.

Henry Ford Health Syst. University Hospital, Toulouse Advantagene, Inc. Silence Therapeutics GmbH

Imperial College London University of Alabama University of Alabama Baxter Healthcare Corporation Spark Therapeutics, LLC St. Jude Child. Res. Hosp. Spark Therapeutics, LLC Alnylam Pharmaceuticals

Fowzan Alkuraya Sanofi University of Oxford

Sponsor

Development phase

Delivered by Ingenta to: SUNY Upstate Medical University IP: 5.8.47.242 On: Fri, 13 May 2016 20:50:34 Copyright: American Scientific Publishers

Country

NCT01574729 NCT00004225 NCT00059605 NCT00126724

NCT00415454 NCT01274455 NCT00638612 NCT01808638

NCT01621867 NCT00004471 NCT00004806 NCT01687608 NCT01620801 NCT00979238 NCT00515710 NCT02035605

China USA USA USA

USA France USA Germany

UK NA NA USA USA, AU USA, UK USA USA

NCT01482195 Saudi Arabia NCT01505062 USA, France NCT01461213 UK

NCT00985517 USA NCT01621581 USA NCT00627588 France, UK NCT00229736 USA

NCT number

Oct 2015 Completed Completed Completed

Terminated∗ Completed May 2013∗∗ Aug 2015

Jun 2014 Completed Completed Nov 2029 Oct 2029 Jul 2029 Aug 2019 Nov 2015

Aug 2023 Jan 2017 Jun 2016

Nov 2014 Jan 2018 Completed Completed

Estimated date of completion

Notes: NTRN—Neurturin; GDNF—Glial cell-derived neurotrophic factor; AADC—Aromatic amino acid decarboxylase TH—Tyrosine hydroxylase; CH1—GTP-cyclohydrolase 1; hMERTK—Human c-mer protooncogene tyrosine kinase; MYO7A—Myosin VIIA; REP1—Rab-escort Protein 1; CFTR—Cystic fibrosis transmembrane conductance regulator; F9—Factor 9; AT—Antithrombin; yCD—Yeast cytosine deaminase; TK—Herpes simplex virus thymidine kinase; ADP—Adenovirus death protein; SST2—Somatostatin receptor subtype 2; DCK—Fusion protein of human deoxycytidine kinase; UMK—Uridine monophosphate kinase; PKN3—Protein kinase N3; TUSC2—Tumor suppressor candidate 2; TNFR:Fc—Tumor Necrosis Factor Receptor: Fc Fusion Protein; AAV2—Adeno-associated virus type 2; LV—Lentivirus; CL—Cationic lipid; LP—Lipoplex; NPh—Neutral phospholipid; AAV8—Adeno-associated virus type 8; GalNAc—Asialoglycoprotein receptor ligand derived from N-acetylgalactosamine; AdV—Adenovirus; PP—Polyplex; PEG—Polyethylene glycol; CED—Convection enhanced delivery; IV—Intravenous; NA—Not available; ∗ Recently terminated due to poor enrolment; ∗∗ Estimated date for primary outcome measure.

Rheumatoid arthritis

Lung cancer

Pancreatic cancer

CL LP CL-NPh LP CL LP AAV8 AAV8 AAV2/8 AAV2 GalNAc

AAV2 LV AAV2

AAV2 AAV2 LV AAV2

Vector

yCD-TK-ADP AdV SST2-DCK-UMK PP TK AdV PKN3 siRNA CL-NPh-PEG LP

hMERTK MYO7A REP1

Retinitis pigmentosa

Hemophilia

NRTN GDNF AADC-TH-CH1 hAADC

Parkinson’s disease

Gene/siRNA

Current clinical trials for gene therapy.

Clinical setting

Table II.

Oliveira et al. Recent Advances in Nucleic Acid-Based Delivery: From Bench to Clinical Trials in Genetic Diseases

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of the potential of this suicide gene therapy to augrecognized to be involved in lung cancer development, ment radiotherapy effectiveness in pre-clinical models. when inactivated. This formulation makes use of cholesThis therapeutic strategy makes use of replicationterol and DOTAP, and was intravenously administered in a competent adenoviruses to deliver a cytosine deaminase phase 1 clinical trial (NCT00059605). Apart from a toxic (CD)/herpes simplex virus thymidine kinase (HSV-1 TK) response upon delivery of the transgene, counteractable fusion gene to tumors carrying two suicide gene syswith pre-medication, results suggest this treatment was tems (CD/5-fluorocytosine and HSV-1 TK/valganciclovir) well tolerated.142 Based on the current biologic treatment for rheumatoid which render malignant cells sensitive to specific pharmaarthritis, some clinical trials have been conducted with the cological agents and sensitize them to radiation allowing intent of blocking inflammatory cytokines responsible for the production of toxic metabolites only inside the tumor, this disease. One of such trials used recombinant adenthereby avoiding systemic toxicity. Apart from mild, tranoviruses to deliver the human TNF receptor immunoglobsient side effects, the treatment was well tolerated in the ulin Fc (TNFR:Fc) fusion gene (NCT00126724). After animal model of the disease.138 Adenovirus delivery of herpes simplex virus thymidine kinase gene (NCT00638612) intra-articular injection patients showed a significant followed by anti-herpetic treatment have all proven to be improvement in target joint global visual analogue scale in safe in pancreatic cancer patients.139 Phase 2 trials will comparison to the placebo group. Overall, treatment was provide further evidence whether they can become cliniwell tolerated except for two serious adverse events and cally relevant in the future. the death of one patient, not correlated with the treatment There is a phase 1 trial in pancreatic cancer that has itself.143 Since the controversy about the death of a young been performed with a PP-based vector (NCT01274455). patient14 and development of leukemia in children subIt is a gene transfer preparation of a pDNA encoding jected to X-linked severe combined immunodeficiency somatostatin receptor subtype 2 (SST2) and a fusion protreatments144 in gene therapy clinical trials, ethics and tein of human deoxycytidine kinase (DCK) and uridine risk acceptability have stood close together. Risk is cermonophosphate kinase (UMK), complexed to a synthetic tainly an inherent part of clinical trials; however the latpolycationic carrier, PEI. Both transgenes induce an antiter serious adverse events were correlated with insertional tumor bystander effect and render gemcitabine chemothermutagenesis145 which enhances the importance of preapy treatment more efficient. The clinical trial is in phase 1 Delivered by Ingenta to: SUNY Upstatestudies Medical University clinical to be as thorough as possible. Correlating and, since last updated on April 2013, no information IP: 5.8.47.242 On: Fri, 13 May 2016 20:50:34 side effects in the animal model with risks humans are about primary outcome, feasibility and safety and second Copyright: American Scientific Publishers going to be incurring in by enrolling in the study may be outcome, antitumoral effect, have been provided. Furtherone of the most relevant actions for risk assessment.146 more, a lipoplex composed of a cationic lipid, a neuFor that reason and under the light of the scarce infortral lipid and a pegylated lipid carrying siRNA against mation available at this point, Table III summarizes the protein kinase N3 has been evaluated in a phase 1 clinmain adverse events encountered in ongoing and recently ical trial for treatment of advanced pancreatic cancer. finished clinical trials. The therapeutic was well tolerated in absence of preIn Parkinson’s disease clinical trials AAV2 delivery of medication and allowed disease stabilization in 52% of NRTN astonishingly did not evidence any adverse events patients after repeated treatment. A phase 1/2 trial for comin the rat and monkey animal model of the disease, binatorial treatment with gemcitabine is currently ongoing even though observation was scrupulous.130 This unavoid(NCT01808638).140 A new set of molecular targets is being introduced in able situation would in fact have exposed patients to an pre-clinical models as potential treatments for lung canunknown risk. Fortunately, phase 1/2a clinical trial only cer, but current clinical trials essentially focus on p53 tarrevealed mild, transient side effects, mostly related to the geted adenoviral delivery. Most recently, a phase 2 trial surgical procedure.130 131 A similar situation occurred in the hemophilia trial, where delivery of F9 using AAV8 (NCT01574729) was supposed to initiate, in China, for resulted in a minor incidence of adverse events registered, evaluation of surgery combined with recombinant adennone severe.147 ovirus encoding p53 gene wound surface injection, for Since both trials used viral vectors, immune responses non-small cell lung carcinoma in comparison with the have to be taken into consideration very seriously. Indeed, surgical procedure and chemotherapy. Similarly, other it is interesting to note that in both situations there was clinical trials have also evaluated the combination of development of antibodies against the viral capsid; howadenoviral p53 delivery together with radiation therapy ever no reactive disease was associated.131 135 (NCT00004225). Three months after completion, results There is no available information so far in the literahave shown this latter approach to be well tolerated, with ture about the performance of adenovirus carrying yCDmain adverse events being fever and chills. Thus, results TK-ADP for the treatment of pancreatic cancer. However, show evidence of efficient tumor regression.141 It is also noteworthy the development of LP carrythe information resultant from pre-clinical trials suggests ing the tumor suppressor candidate 2 (TUSC2), a gene a minor immune reaction to the carrier, which should 852


Oliveira et al. Table III.


Pre-clinical and clinical safety evaluation of vectors for nucleic acid based therapy. Adverse events Pre-clinical

Clinical setting Parkinson’s disease

Vector type Viral

Gene/ DNA

Immune stimulation

NRTN

∗

Clinical Immune stimulation

Other

Anti-AAV2 Ab

No toxicity

Anti-AAV2 Ab

∗

Other Hallucination or dyskinesia post-surgical symptoms ↑T2 MRI signal

Hemophilia

F9

Anti-AAV8 Ab ↑Neutralizing Ab∗

No toxicity

Primary immune response to AAV8

Anemia Bradycardia

Pancreatic cancer

TK

Neutrophilia

Acute pancreatitis ↑Amylase ↑Liver inflammation

NA

Azotemia Dehydration ↑Bilirubin ↑AST

NA

NA

↑Neutrophilia ↑C-reactive protein ↑Interleukin-6 ↑Total white-cell count

Influenza-like symptoms ↑↑Airway symptoms ↓Forced expiratory volume

Cystic fibrosis

Non-viral

CFTR

Notes: NRTN—Neurturin; F9—Factor 9; TK—Herpes simplex virus thymidine kinase; CFTR—Cystic fibrosis transmembrane conductance regulator; AAV2— Adeno-associated virus type 2; Ab—Antibodies; AAV8—Adeno-associated virus type 8; ∗ —No associated reaction; NA—Not available; MRI—Magnetic resonance imaging; AST—Aspartate aminotransferase; Severity scale: ↑—minor; ↑↑—mild; ↑↑↑—strong; ↓—decreased.

be valued in further studies.138 Furthermore, the clinnucleic acids by extracellular enzymes that reduce the amount of gene cargo available to reach the target cell. ical trial combining delivery of HSV-thymidine kinase The formulation of stable LP and PP-nucleic acid gene and valganciclovir, together with 5-fluorouracil based vectors that claim the chemical and biophysical properchemoradiation in patients with advanced pancreatic canDelivered by Ingenta to: SUNYties Upstate Medical University required to overcome extracellular barriers has been cer, demonstrated adverse events to be transient symptoms IP: 5.8.47.242 On: Fri, 13described May 2016 20:50:34 elsewhere.149 Potentially rate-limiting barriers of azotemia, dehydration and elevation of bilirubin and Copyright: American Scientific Publishers have been disclosed by using high-resolution fluorescence aspartate aminotransferase levels.139 microscopy and live cell confocal microscopy in combiConversely, clinical trials involving non-viral delivery nation with various probe-labeling technologies that visuof the CFTR gene revealed both immune stimulation and alize co-localization of nucleic acids with intracellular respiratory symptoms which were probably related to the organelles.150 151 pro-inflammatory action of the lipid that may be processed Considering the number of research groups that focus by macrophages. Furthermore, because of the potential their investigations on the development of new vectors for of methylated CpG dinucleotides to trigger inflammatory nucleic acids therapy, together with the advances in the responses upon recognition as foreign by humans,134 CpGdevelopment of new technologies to better understand their free optimized plasmids97 are now being used in current intracellular traffic, the present limitations of non-viral clinical trials. vectors will be resolved rationally. Thus, rational design Concomitantly, it is of the utmost importance, not to of non-viral vectors mimicking viral vectors would be a neglect non-viral vectors themselves, which can as well way to break this bottleneck. cause deleterious effects. Hence, careful consideration Accordingly, and to our current knowledge, the most has to be put on structure-activity relationship to minipromising non-viral vectors for siRNA and pDNA delivmize lipid or polymer induced toxicity during pre-clinical 148 ery stand on this premise. PP are advantageous for nucleic trials. acid delivery because they are usually less immunogenic than their lipidic counterparts and easy to manipulate and CHALLENGES FOR SUCCESSFUL CLINICAL functionalize, which has incited the use of a wide range APPLICATION OF NON-VIRAL VECTORS of polymers;152 however transfection efficiencies often do Clinical application of non-viral vectors has been hamnot meet the expectations and have hampered their propered by their transfection efficiencies, largely due to the gression in clinical trials. In view of this, optimization of various extracellular and intracellular barriers that impede PP evidenced by structure-activity relationship studies has their performance. Nucleic acids delivery has resulted in greatly contributed for their improvement. Since biopolynumerous studies but has produced a distinct shortage of mers can be extensively and precisely engineered, research licensed clinical treatments for genetic diseases. Shortcomefforts have been devoted to improve natural polymers ings have arisen due to the degradation of unprotected with greater intracellular trafficking.153 Complementary, J. Biomed. Nanotechnol. 12, 841–862, 2016

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insights into the dynamics of the physicochemical properties of the polymer on the encapsulation efficiency of nucleic acids,154 have elucidated strategies for the development of more successful PP. Furthermore, taking into consideration the impact hydrodynamic size has on differential activation of cell internalization pathways,155 the ability to obtain PP in the nanoscale making use of biopolymers has been a significant achievement for their potential as nonviral vectors.156 Chitosan is a natural occurring polymer that epitomizes all the aforementioned properties157 whilst it provides as well tunable electrostatic binding to negatively charged nucleic acids giving rise to PP. As depicted in Figure 3, the cationic surface charge resultant from PP assembly allows for efficient cellular uptake due to the anionic charge of the cell membrane, thereby inducing endocytosis.158 Upon endosomal entrapment, the high buffering capacity of chitosan in the endolysosomal pH range enables the induction of the proton sponge effect as compared to PEI, while deprived of the cytotoxicity associated with the latter.159 The physicochemical properties of the polymer greatly impact on its potential as a non-viral vector160 and therefore have been subject to extensive optimization. Altogether, it has been described that the most efficient chitosan PP reflect these properties on being endowed with a subtle balance between PP stability for nucleic

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acid protection though preserving the ability to dissociate in coordination with lysosomal escape.161 Congruently, optimized formulations were successful in mediating the delivery of both pDNA and siRNA in diverse clinical settings162163 as well as in achieving outstanding hydrodynamic sizes under 5 nm for brain-targeted delivery.80 164 Conversely, rational strategies of structure modification have conferred similar properties to synthetic polymers, increasing their biocompatibility and providing satisfying results, as is the case of poly(beta-amino esters).165 The latter have been carefully optimized, in a high-throughput fashion, to overcome specific barriers of cell transfection, as represented in Figure 4. In view of that, it was described that small molecule end-group modifications are critical for cellular uptake of these polymers, internalization being favored for amine monomer end-capped structures in a way largely independent of other physicochemical properties. Secondly, the reversibly protonated secondary and tertiary amines of these polymers allow for endosomal escape through the proton sponge effect, similarly to other polymers.63 Moreover, upon successful release into the cytoplasm, nucleic acid dissociation occurs through the hydrolytic cleavage of the esters linkages of the polymer,

Delivered by Ingenta to: SUNY Upstate Medical University IP: 5.8.47.242 On: Fri, 13 May 2016 20:50:34 Copyright: American Scientific Publishers

Figure 3. The cationic surface charge of chitosan polyplex (PP) facilitates the establishment of interactions with the oppositely charged cell membrane and triggers the endocytic uptake of PP (A). The acidic environment of endolysosomes is buffered by the amine groups of the polymer, a mechanism referred to as the proton sponge effect (B). This leads to osmotic swelling and culminates in endolysosomal burst, consequently releasing PP into the cytoplasm (C). The physicochemical properties of the polymer determine the efficient separation of the polymer and nucleic acids (D), which will proceed to exert its action together with the intracellular machinery.

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Figure 4. Structural modifications of poly(beta-amino esters) allowed for optimization of polyplexes by conferring them the ability to overcome cell uptake barriers. The internalization process is favored for amine end-capped polymers as opposed to their acrylate counterparts (A); Tertiary and secondary amines induce the proton sponge effect inside endolysosomes (B) and lead to nucleic acid release into the cytoplasm where the ester linkage of poly(beta-amino esters) is cleaved (C) conferring degradability to the polymer. J. Biomed. Nanotechnol. 12, 841–862, 2016

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conferring them an advantage over other formulations by surpassing a difficulty created due to the strong interactions established during PP formation.166 These structural properties were designed to surpass the major bottlenecks of intracellular delivery that have been previously identified, and led to the selection of the lead structures for improved results. Still, poly(beta-amino esters) exhibit cytotoxic effects that present an engineering challenge167 and more comprehensive pre-clinical investigations such as exact quality control of polymers, pharmacokinetics and toxicological studies should be performed.168 Poly(beta-amino esters) have become very relevant for PP preparation. Nevertheless, although positive results have been accomplished for delivery of both pDNA and siRNA, the previous represents a bigger challenge requiring a high concentration of polymer for better particle formation and siRNA complexation.169 Knockdown was determined to improve with increasing molecular weight of the polymer and hydrophobicity.170 Stable nucleic acid lipid particles (SNALP) are rationally designed LP that were developed from the selection of the best performing ionizable lipid amongst a Figure 5. SNALP delivery is an apoE dependent mechanism combinatorial formulation. This component plays a dual that allows efficient targeting to hepatocytes. The neutral surrole in the delivery process since it facilitates the estabface charge of lipoplexes in the blood compartment leads to the exchange of apoE which binds to cell receptors at the surlishment of electrostatic interactions with nucleic acids face of hepatocytes and allows for endocytic uptake. Thereinto nanoparticles as well as contact with the cell memafter, upon acidification of the endosomal compartment, the 171 Furthermore, brane that allows for endocytic uptake. ionizable acquires a positive charge that mediates the Delivered by Ingenta to: SUNY Upstate lipid Medical University the potential of SNALPs stands on the knowledge of the fusion between the latter 5.8.47.242 On: Fri, 13 May 2016 20:50:34 and the endosomal anionic memdynamics of endosomal membrane IP: destabilization, which brane inducing its destabilization. Thus, efficient release of Copyright: American Scientific Publishers is hypothesized to rely on the properties of the ionizable nucleic acids into the cytoplasm occurs. lipid headgroups that, when protonated and upon fusion with the endosomal membrane anionic lipids, enable adopthere is no guarantee of efficient delivery for tissues tion of an inverted non-bilayer structure. In view of other than the liver. Thus, further strategies prove to be this, smaller phospholipid headgroups, when compared relevant. to the sum of the headgroup area of the cationic and Macrocyclic platforms with potential for the incorpoanionic lipid species isolated, are favorable to enable this ration of functional elements in a well-defined architec172 configuration. Furthermore, systematic structural modture have emerged as interesting alternatives.176 Increasing ifications to hydrophilic headgroups allowed for modulamultivalency of non-viral vectors has led to more specific, tion of the chemical properties that determine the surface thermodynamic and kinetically more stable interactions.177 charge of the nanoparticles in a pH-dependent manner. These organized architectures are still in pre-clinical phase, The latter have been found to be fundamental for the but they allow the establishment of flexible and cooperasuccess of the delivery system.173 Overall, in the blood tive interactions which enhance a biomimetic behavior.178 compartment SNALPs adopt a neutral surface charge, One of such systems are calixarenes, which have been used determinant for minimizing cytotoxicity, in addition to in different formulations for nucleic acid delivery such as acquiring apolipoprotein E (apoE), which is known to solid lipid nanoparticles.179 Their conical architecture comassociate with neutral membrane surfaces, and allows bined with functionalization of the upper and lower rim for targeted delivery to hepatocytes through binding with are thought to enable various reactive positions and selfcell surface receptors mediating endocytic uptake. Thereassembling into structures of nanometric scale, able to act after, endosomal acidification renders SNALPs positively as carriers through membranes.180 charged which induces the combination of the cationic Cyclodextrins are cyclic oligosaccharides composed lipids with the anionic endosomal membrane, leading to of -(1-4)-linked D-glucopyranose units with a basketits disruption and release of nucleic acids into the cytolike structure, able to retain hydrophobic compounds plasm, as demonstrated in Figure 5.174 This behavior has in the inner core and hydrophilic compounds on the proven extremely successful for hepatic delivery of siRNA, exterior.181 Low toxicity, high stability and the ability as demonstrated in recent clinical trials,175 and may represent the approach closest to clinical application. However, of chemical modification render them useful for nucleic J. Biomed. Nanotechnol. 12, 841–862, 2016

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acid-based delivery. Similarly to calixarenes, functionalized amphiphilic cyclodextrins have also been described to self-assemble into nanocomplexes of 40–80 nm diameter, promoting the condensation of DNA and an efficient transfection of cells.182 Interestingly, a resemblance in shape and charge can be observed for both non-viral vectors hence one might infer about the importance of the complex architecture of these systems for delivery of nucleic acids. In view of that, Figure 6 illustrates an approach for the mechanism of DNA condensation and delivery with formulations made of amphiphilic cyclodextrins and calixarenes.182 183 Studies have shown that calixarenes enter the cell through a nonendocytic pathway, possibly through a plasma membrane carrier, and experience a rapid uptake process.184 Alternatively, cyclodextrins are internalized via a non-endocytic pathway185 whose process of endosomal escape is suggested to rely on similar mechanisms to those attributed to LP and PP.186 Studies on internalization mechanisms may elucidate new strategies for functionalization of macrocyclic structures and potentiate even further cellular uptake.186 Altogether, these characteristics undoubtedly benefit their development as non-viral vectors and project an auspicious line of research.

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CONCLUSIONS

The advances in genetics and biology of disease have been remarkable during the last two decades, nevertheless few innovative and satisfying medicines have increased the therapeutic arsenals in the 21st century. Successful gene therapy is a long expected scientific revolution but the reality between recent laboratory progresses contrast strongly with what actually reaches clinical application. In clinical settings such as retinitis pigmentosa and rheumatoid arthritis few clinical trials are currently registered, whereas in Parkinson’s disease and hemophilia the majority makes use primarily of viral vectors, which do not make justice to the multiplicity of different carriers that have been developed. It is, thus, urgent to narrow down the fields of disease biology and nanotechnology to accelerate the development of new medicines. Whilst presenting a much improved safety profile, one of the major impediments associated with the use of nonviral vectors is their poor cell transfection properties, a tendency that has been countered with development of new strategies such as optimization of polymers and lipids in formulations, surface modifications and coupling of targeting ligands. The evaluation of multiple parameters associated with barriers to delivery such as nucleic acids entrapment, pKa, vector stability, and cell uptake as a collective may serve as a useful prescreening tool for the of non-viral vectors in vivo. Delivered by Ingenta to: SUNY advancement Upstate Medical University Multiparametric approaches have been the basis for the IP: 5.8.47.242 On: Fri, 13 May 2016 20:50:34 development of several delivery systems rationally optiCopyright: American Scientific Publishers mized and designed to overcome the main transfectionlimiting barriers. Poly(beta-amino esters) and chitosan are amongst the latter and have provided very encouraging results, whilst SNALPs have successfully proven clinical translatability. Macrocyclic multivalent systems, amphiphilic calixarenes and cyclodextrins, stand as one of the most promising approaches since they allow for high transfection efficiency along with very little toxicity, instigating further research. Promising results have also been retrieved from recent clinical trials, namely for Parkinson’s disease, which brings nucleic acid-based delivery closer to clinical application and offers great hope in managing diseases. Even though no major adverse events have been encountered, a thorough monitorization is fundamental to prevent serious immune responses, as novel non-viral vectors pave their way to clinical trials. Figure 6. Nucleic acid condensation by calixarenes (A) and cyclodextrins (B) which aggregate in the form of multivalent nanostructures. Cyclodextrins nanocomplexes (C) have been described to enter the cell through an endocytic pathway which leads to their entrapment inside endosomes (D). Escape from these cellular compartments is crucial for efficient transfection of cells (E) and appear to rely on mechanisms similar to those of lipo- and polyplexes. Alternatively, calixarenes nanoparticles (F), resulting from the assembly of several nanomicelles (G) in the presence of nucleic acids, are suggested to be internalized through a plasma membrane carrier (H).

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