Liposomal delivery of nucleic acid-based anticancer therapeutics: BP-100-1.01.

Review

Liposomal delivery of nucleic acid-based anticancer therapeutics: BP-100-1.01 1.

Introduction

2.

Chemical modifications of nucleic acids

3.

Intracellular delivery of nucleic

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acids 4.

BP-100-1.01

5.

Conclusion

6.

Expert opinion

Ana Tari Ashizawa & Jorge Cortes† †

University of Texas MD Anderson Cancer Center, Department of Leukemia, Houston, TX, USA

Introduction: Antisense oligonucleotides, siRNA, anti-microRNA are designed to selectively bind to target mRNAs, and silence disease-causing or -associated proteins. The clinical development of nucleic acid drugs has been limited by their poor bioavailability. Areas covered: This review article examines the strategies that have been utilized to improve the bioavailability of nucleic acids. The chemical modifications made to nucleic acids that have improved their resistance against nuclease degradation are briefly discussed. The design of cationic and neutral lipid nanoparticles that enable the systemic delivery of nucleic acids in vivo is reviewed, and the proof-of-concept evidence that intravenous administration of nucleic acids incorporated into lipid nanoparticles leads to decreased expression of target genes in humans. Preclinical results of the neutral BP-100-1.01 nanoparticle are highlighted. Expert opinion: To further improve the clinical potential of nucleic acid cancer drugs, we predict research on the next generation of lipid nanoparticles will focus on: i) enhancing nucleic acid delivery to poorly vascularized tumors, as well as tumors behind the blood--brain barrier; and ii) improving the accessibility of nucleic acids to the cytoplasm by enhancing endosomal escape of nucleic acids and/or reducing exocytosis of nucleic acids to the external milieu. Keywords: anti-microRNA, antisense oligonucleotides, cationic liposomes, dioleoyl phosphatidylcholine, drug delivery, gene silencing, growth factor receptor bound protein-2, neutral liposomes, siRNA Expert Opin. Drug Deliv. [Early Online]

1.

Introduction

Nucleic acids such as antisense oligodeoxynucleotides (oligos), anti-microRNA (anti-miR) and siRNA are used as RNA interfering molecules to repress gene expression. These nucleic acid-based therapeutics, typically in the range 15 -- 21 bases in length, bind to their target RNAs with perfect base-pairing. These molecules have tremendous therapeutic potential, especially in silencing disease-causing or disease-associated genes that are not amenable to conventional therapeutics such as small molecules or monoclonal antibodies. Antisense oligos and anti-miRs are single-stranded molecules, while siRNAs are double-stranded molecules. Antisense oligos and siRNAs are typically used to target the coding regions of the mRNA, though antisense oligos have been used to target the 5¢ and the 3¢ untranslated regions (UTR) of the mRNA. Anti-miRs target microRNAs (miR), which bind to the 3¢ UTR of the mRNA. The therapeutic potential of nucleic acids has been investigated in numerous preclinical models and clinical trials. However, antisense oligo is the only nucleic acid that has been approved by the US FDA as therapeutic modality. The antisense drugs fomivirsen (marketed as Vitravene) and mipomersen (marketed as Kynamro) were approved in August 1998 and January 2013, respectively. Fomivirsen is used as a 10.1517/17425247.2015.996545 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted

1

A. T. Ashizawa & J. Cortes

Article highlights. . .

.

Clinical development of nucleic acids has been limited by their poor bioavailability. Cationic as well as neutral lipid nanoparticles have been developed to deliver nucleic acids systemically. Clinical trial data reveals that lipid nanoparticles enable nucleic acid drugs (antisense oligos or siRNAs) to reach their target sites, when administered intravenously. Future development of lipid nanoparticles should focus on enhancing the accumulation/retention of nucleic acids in the cytoplasm, and on delivering nucleic acids to poorly vascularized tumors as well as to tumors within the CNS.


This box summarizes key points contained in the article.

2.

treatment for cytomegalovirus retinitis, while Kynamro is used as a treatment for homozygous familial hypercholesterolemia. siRNAs induce gene silencing by utilizing the RNA-induced silencing complex (RISC) to cleave and degrade target mRNAs. siRNAs are generated from the cleavage of long doublestranded RNA molecules by Dicer, an endoribonuclease [1,2]. Upon being loaded into RISC, the sense (complementary) siRNA strand is degraded and the antisense (mature) siRNA guides the interaction of the siRNA--RISC complex with target mRNA. Argonaute 2, an endoribonuclease within the RISC complex, cleaves the target mRNA leading to mRNA degradation. miRs are transcribed by RNA polymerase II, which acts on the DNA and releases primary-miRs [1,3]. Primary-miRs are processed by the RNaseIII enzyme Drosha to yield pre-miRs, which are 70-nucleotide-long hairpin structures. The pre-miRs are then cleaved by Dicer to form miRs. Similar to siRNAs, miRs are loaded into RISC, where the sense strand is degraded and the seed sequence (the initial 6 -- 8 nucleotides from the 5¢ end) of the antisense strand guides the interaction of the miR--RISC complexes with target mRNA. Perfect base pairing of the miR to the target mRNA results in mRNA degradation, whereas imperfect base pairing leads to translational repression of the target protein. Numerous miRs are overexpressed in cancer cells, resulting in gene deregulation and cancer progression. AntimiRs are designed to bind to miRs and repress miR activities. Antisense oligos do not utilize the RISC complex. Instead, the mechanisms by which antisense oligos induce gene silencing are: i) activation of RNase H that leads to degradation of target mRNAs; and ii) steric hindrance that blocks ribosomes from binding to and translating target mRNAs [4]. RNase H is activated when antisense DNA drugs bind to target mRNAs, forming DNA--RNA hybrids. Upon activation, RNase H cleaves target mRNAs, which are then degraded by exonucleases. However, many antisense oligo analogs cannot activate RNase H; in these instances, the mechanism of gene silencing is steric hindrance. RNase H activation is often a desirable mechanism of antisense drugs because in theory, gene silencing resulting from 2

mRNA degradation should be more potent than steric hindrance. Nonetheless, the therapeutic potential of antisense drugs that invoke the steric hindrance mechanism has been actively explored in some neurodegenerative diseases to modulate gene splicing [5]. Pre-mRNA splicing is the process by which specific intronic and some exonic sequences that are not required for mature mRNA generation are removed. Mutations in the dystrophin gene cause the gene to be misspliced and produce an unstable dystrophin transcript. Through sequence-specific hybridization to the pre-mRNA, antisense oligos act as steric blocks and alter the mis-splicing of the mutated dystrophin transcript.

Chemical modifications of nucleic acids

Much research was undertaken to improve the pharmaceutical development of antisense oligos, as only a small fraction of the injected antisense dose reaches the cytoplasm of target cells [6]. Natural phosphodiester DNAs are susceptible to nuclease degradation. To increase the resistance of antisense oligos against nuclease degradation, chemical modifications were made in the phosphate backbone or the deoxyribose/ribose sugar ring of nucleic acids. Backbone modifications Backbone modifications are made in the inter-nucleotides phosphate bridges. 2.1

. The phosphorothioate modification replaces an oxygen

atom of the phosphate group with a sulfur atom. The Vitravene antisense drug is a 21-base phosphorothioate oligo (Table 1). . The P-ethoxy modification adds an ethyl group to one of the nonbridging oxygen atoms in the phosphate group. BP-100-1.01, a Phase I investigational drug, is an 18-base P-ethoxy oligo (Table 1). Both phosphorothioates and P-ethoxys are more stable in serum than phosphodiesters because they are resistant to nuclease degradation. Phosphorothioates, like phosphodiesters, are negatively charged but P-ethoxys are uncharged. 2.2

Sugar modifications . Several modifications have been made in the deoxyri-

bose or ribose sugar ring, including morpholinos, locked nucleic acids, 2’-O-methyl RNA and 2’-methoxy-Oethyl RNA. . Morpholinos have standard nucleic acid bases. But their bases are bound to morpholine rings instead of deoxyribose rings. The bases are linked through phosphorodiamidate groups, not phosphates. AVI-4658 and AVI-7100 are morpholino antisense oligos currently being studied in clinical trials (Table 1).

Expert Opin. Drug Deliv. (2014) 12(7)

Liposomal delivery of nucleic acid-based anticancer therapeutics: BP-100-1.01


Table 1. Antisense or anti-miR drugs that are FDA-approved or currently in clinical trials. Nuclei acid modification

Drug*

Targeted gene

Clinical status

Indications

Backbone modifications Phosphorothioatez

Vitravene

Approved

Cytomegalovirus retinitis

P-ethoxy

BP-100-1.01

Cytomegalovirus replication major immediate early region 2 Growth factor receptor bound protein-2

Phase I

Leukemia

Sugar modifications Morpholino Morpholino Locked nucleic acid Locked nucleic acid 2’-O-methyl 2’-methoxy-O-ethyl

Eteplirsen AVI-7100 SPC2968 Miravirsen* Drisapersen Mipomersen

dystrophin exon 51 Influenza virus Hypoxia inducing factor-1a miR-122 dystrophin exon 51 Apolipoprotein B

Phase III Phase I Phase II Phase II Phase III Approved

Duchenne myotonic dystrophy Influenza Hepatocellular carcinoma Hepatitis C Duchenne myotonic dystrophy Homozygous familial hypercholesterolemia

*All the drugs listed above are antisense oligos, except Miravirsen which is an anti-miR. z Phosphorothioate is the only analogue that can recruit RNase H.

. A locked nucleic acid is modified with a bridge connect-

ing the 2’ oxygen and 4’ carbon of the ribose sugar. The bridge ‘locks’ the ribose in the 3’-endo conformation. SPC2968 and Miravirsen are locked nucleic acid antisense drugs, which are in clinical trials (Table 1). . 2’-O-methyl RNA has a methyl group added to the 2’ hydroxyl group on the ribose ring. Drisapersen, a 2’O-methyl analog, was investigated in a Phase III trial (Table 1). . 2’-methoxy-O-ethyl RNA has an ethyl group that connects the 2’ hydroxyl group on the ribose sugar to a methoxy group. The approved drug, Mipomersen, has 2’-methoxy-O-ethyl substitutions (Table 1). All these sugar-modified analogs have longer serum half-lives than phosphodiesters because they are resistant to nuclease degradation. Locked nucleic acids, 2’-O-methyl RNA, and 2’-methoxy-O-ethyl RNA are negatively charged. However, morpholinos are uncharged because the negatively charged phosphates are replaced with uncharged phosphorodiamidate groups. Gapmers Steric block-acting antisense oligos tend to be more tolerant of chemical modifications than RNase H activating-antisense oligos. This is probably because extensive chemical modification prevents RNase H from recognizing and binding to the antisense oligo. Phosphodiesters and phosphorothioates are the only antisense that have the ability to activate RNase H [7]. But antisense oligos made entirely of phosphorothioates do not bind to target mRNAs as tightly as their phosphodiester counterparts [7]. It was estimated that the melting temperature of a phosphorothioate--RNA duplex is approximately 0.5 C per nucleotide lower than a phosphodiester--RNA duplex [4]. A chimeric antisense oligo incorporating both nuclease resistance and RNase H activation characteristics has appeared in the form of a ‘gapmer.’ 2.3

A gapmer contains a central block of phosphodiester or phosphorothioate DNA nucleotides, which is flanked by ‘wings’ or blocks of modified nucleotides composed of locked nucleic acid, 2’-O-methyl, or 2’-methoxy-O-ethyl at the ends. The central block of phosphodiester or phosphorothioate DNA nucleotides is designed to activate RNase H to degrade target mRNA, while the ‘wings’ are designed to protect the central DNA block from nuclease degradation and to ensure tight binding between the antisense oligo and its target. Using the same oligo sequence, Grunweller et al. [8] reported that a locked nucleic acid gapmer, composed of a central phosphodiester DNA block flanked by locked nucleic acid wings, was about 175-fold more potent than a full-length phosphorothioate antisense oligo in suppressing the expression of vanilloid receptor subtype 1, which is a transient receptor potential family member essential for pain perception. Although phosphorothioates are stable in serum, they have been found to induce toxicity in vivo. Primates treated with phosphorothioates were found to have the complement cascade transiently activated, which in some cases led to cardiovascular collapse and death [7]. Also, primates treated with phosphorothioates had altered clotting cascade [7]. The mechanism by which phosphorothioates induce in vivo toxicity is not clear, but may be related to their binding to proteins like heparin-binding proteins that interact with polyanions [7]. Interestingly, modifying the wings of a phosphorothioate oligo lowers its toxicity. Agrawal [9] demonstrated that substituting the wings of a phosphorothioate with four 2’-O-methyl RNA nucleosides at each end blocked platelet decrease as well as attenuated increases in serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) levels, which are indicators of hepatic toxicity. The Mipomersen antisense drug is a gapmer composed of a central block of 10 phosphorothioate linkages flanked by five 2’-methoxy-O-ethyl nucleosides on each end. Anti-miRs and siRNAs face the same poor bioavailability challenge as antisense oligos. Chemical modifications improve


3



the serum stability and the potency of anti-miRs. AntimiR-122 composed of locked nucleic acid-modified oligo (SPC3649) was intravenously injected into chimpanzees that have chronic hepatitis C virus infection. SPC3649 was administered at a dose of 5 mg/kg on a weekly basis for 12 weeks. A decrease of 2.3 orders of magnitude in hepatitis C virus RNA levels was found in the livers of animals treated with SPC3649 [10]. Chemical modifications also improve the serum stability of siRNAs. However, the modifications could reduce siRNA potency. A single 2’-O-methyl modification at position 14 of the siRNA antisense strand substantially reduced its gene silencing activity. The reduced gene silencing activity was due to decreased loading of the modified siRNA antisense strand into RISC and lower silencing potency of the modified siRNA antisense strand [11]. Fluorochromes conjugated to the 5¢ or the 3¢ end of the siRNA sense strand or to the 5¢ end of the siRNA antisense strand did not affect the siRNA gene silencing potency. But fluorochromes conjugated to the 3¢ end of the siRNA antisense strand abolished its gene silencing activity [12]. Modification of siRNA with cholesterol at the 3¢ end of the siRNA sense strand did not affect siRNA gene silencing potency; however, the same modification at the antisense strand significantly reduced its silencing potency [13]. These studies indicate that in general, the siRNA sense strand is more tolerant of chemical modifications than the siRNA antisense strand. In some cases, although modifications do not affect gene silencing activity, they induce toxicity. Phosphorothioate modification of siRNA did not significantly affect gene silencing activity, but cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by a phosphorothioate [12]. On the other hand, modifications like locked nucleic acid or 2’-O-methyl phosphorodithoates, could improve the serum stability of siRNA without compromising its gene silencing potency. Modification of siRNA with locked nuclei acid enhanced siRNA serum stability as well as reduced siRNA off-target effects. Locked nuclei acid modified siRNAs targeting the severe acute respiratory syndrome virus were shown to have improved efficiency over unmodified siRNA [14]. Wu et al. [15] demonstrated that substituting two residues at the 3¢ end of the siRNA sense strand with 2’-O-methyl phosphorodithoates enhanced the loading of siRNAs into RISC, which led to sixfold higher potency than unmodified siRNA in suppressing target protein expression. These studies indicate that the design of an effective nucleic acid-based therapeutic should incorporate a specific target sequence, and dependent on the anticipated mechanism of action, the type and extent of chemical modification.

3.

Intracellular delivery of nucleic acids

Regardless of whether the chemical modifications are made in the backbone or in the sugar ring, nucleic acids do not penetrate into cells easily. Chemical conjugations at the 5¢ or 3¢ 4

terminus as well as delivery vehicles are being investigated to increase their cellular penetrance. Terminal conjugations of nucleic acids Nucleic acids have been conjugated to cholesterol or cellpenetrating peptides (CPPs) at the 5¢ or the 3¢ terminus. Cholesterol is a lipid molecule vital in regulating the fluidity of cell membranes. By being conjugated to nucleic acids, cholesterol enhances the binding of nucleic acids to low-density lipoproteins, leading to improved cellular uptake and efficacy of antisense oligos and anti-miRs [16-18]. Mice bearing hepatocellular carcinoma xenografts had increased survival, when intravenously injected with cholesterol conjugated antimiR-221 compared to unmodified anti-miR-221, because of decreased hepatocellular carcinoma proliferation and increased markers of apoptosis and cell cycle arrest [18]. The cholesterol conjugation enables high uptake of the anti-miR by the hepatocellular carcinoma cells because these cells have abundant low-density lipoprotein receptors. CPPs are short peptides that facilitate cellular uptake of various molecular cargo, including nucleic acids. CPPs could be polycationic, amphipathic or hydrophobic. The mechanisms by which CPPs enhance intracellular delivery of nucleic acids are by direct penetration into cellular membranes or endocytosis [19-21]. Nucleic acids have been conjugated to CPPs, like the HIV 1-derived transactivator of transcription (TAT) peptide [22], the galanin neuropeptide-derived transportan peptide [23] and the drosophila antennapedia proteinderived penetratin peptide [24]. However, direct electrostatic interaction of cationic CPPs with negatively charged nucleic acids could negate the penetrating activity of cationic CPPs. Therefore cationic CPPs should be conjugated to neutral nucleic acids like morpholinos, locked nucleic acids and P-ethoxy oligos. [25].. Uptake, intracellular localization, cytotoxicity and biological activity of siRNAs were significantly dependent on the kind of CPP used and the length of the cationic peptides in the conjugate. Transportan-conjugated siRNAs yielded both high internalization of siRNA and strong gene silencing activity, while penetratin- and TAT-conjugated siRNAs did not [26]. Generally terminal conjugations are amendable to nucleic acids if they are utilized as steric blocks. But these conjugations could hamper RNase H activation or RISC loading. To that effect, nanoparticles designed to protect nucleic acids, specifically siRNA, from nuclease degradation and to enhance their cellular uptake have been developed. 3.1

Lipid-mediated delivery of nucleic acids Liposomes are lipid vesicles composed of an aqueous core compartment enclosed by one or more concentric layers of lipid bilayers. Liposomes could be prepared from one or more types of biodegradable lipids like phosphatidylcholine, cholesterol, sphingomyelin and glycerophospholipids. Since liposomes contain aqueous and hydrophobic compartments, they can encapsulate negatively charged as well as uncharged 3.2



nucleic acids. In our experience using the lyophilization method, liposomes that do not contain cationic lipids typically incorporate negatively charged nucleic acids at £ 70% efficiency. Our lyophilization method involves mixing nucleic acids with phospholipids and the nonionic surfactant Tween 20 in the presence of excess tertiary butanol, before freezing and lyophilizing the mixture. Several studies have included cationic lipids in their preparation of liposomes or lipid complexes because of their ease in binding to negatively charged nucleic acids. Cationic lipid-based nanoparticles The first generation of cationic lipids includes lipofectin and lipofectamine. Lipofectin consists of the cationic lipid N-[I-(2,3,-dioleyloxy)propyl]-N,N,N,-trimethylammonium chloride (DOTMA) and a helper lipid dioleoyl phosphatidylethanolamine (DOPE). DOPE is used to ‘help’ the nucleic acid cargo gain access into the cytoplasm, where it functions. Lipofectamine consists of the polycationic lipid (2,3-dioleyloxyN-(2(sperminecarboxamido)ethyl)-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOPSA) and DOPE. Another example is the diester analog of DOTMA, 1,2-dioleoyl-3trimethylammonium propane chloride (DOTAP). These cationic lipid complexes have poor stability in serum, tend to form large aggregates and are toxic. Some of these toxicities include complement activation, induction of pro-inflammatory cytokines, leukopenia and thrombocytopenia [27]. The toxicities are mainly due to the interaction of the cationic lipid with cell membranes and/or serum proteins. Therefore, the newer generation of cationic lipid formulations, like stable nucleic acid lipid particles (SNALPs), lipidoid nanoparticles, liposomepolycation-hyaluronic acid (LPH), contain an outer ‘coat or shell’ in their formulations to prevent direct interaction of these cationic lipids with cellular membranes and proteins.


3.2.1

Stable nucleic acid lipid particles SNALPs have been used to deliver siRNAs in vivo. In SNALPs, ionizable cationic lipids with pKa values below 7, such as 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLin-DMA), are used to encapsulate siRNAs [28]. At low pH values (e.g., pH 4), the ionizable lipids are positively charged, allowing them to bind to the negatively charged siRNAs. SNALPs are composed of an ionizable cationic lipid, phosphatidylcholine and cholesterol [28]. The cationic lipid/ phosphatidylcholine/cholesterol mixture is dissolved in ethanol and added dropwise to citrate buffer, pH 4.0, to form multi-lamellar vesicles. The multi-lamellar vesicles are extruded to form large unilamellar vesicles. siRNA is added to large unilamellar vesicles with constant mixing, followed by dialysis against phosphate buffered saline, pH 7.4, to remove ethanol and neutralize the pH of the SNALPs. PEG is added to the siRNA/cationic lipid mixture to form a hydration shell around the lipids to provide stability and to evade immune responses. 3.2.2

A screening process, starting with the cationic lipid DLin-DMA as a benchmark, identified 1,2-dilinoleyl4-(2-dimethylaminoethyl)- [1,3]-dioxolane (DLin-KC2-DMA) as the better-performing ionizable lipid [29]. SNALP composed of DLin-KC2-DMA (KC2-SNALP) was demonstrated to have in vivo activity against serum Factor VII (FVII), a hepatic gene, at doses as low as 0.01 mg/kg in mouse models [29]. Additionally, KC2-SNALP was effective in delivering siRNA against transthyretin (siTTR) in nonhumans. Cynomolgus monkeys were administered with a single 15-min intravenous infusion of KC2-SNALP-formulated siTTR at doses of 0.03, 0.1, 0.3 and 1 mg/kg. Efficacy of KC2-SNALP-formulated siTTR was observed at 0.1 mg/kg in cynomolgus monkeys [29]. After additional structural modification to the DLinKC2-DMA lipid, a more potent ionizable lipid DLin-MC3-DMA was identified [30]. DLin-MC3-DMA SNALP-formulated siTTR [30] was approximately 30-fold more potent than KC2-SNALP-formulated siTTR in nonhumans [29,30]. MC3-SNALP had been used to deliver: i) an siRNA against PCSK9 as a cholesterol lowering therapy in a Phase I clinical trial in subjects with hypercholesterolemia [31]; and ii) an siTTR in healthy volunteers in a Phase I trial [32]. Lipidoid nanoparticles Lipidoid particles utilize lipid-like molecules, cholesterol and PEG for the delivery of siRNAs and anti-miRs [33]. The library of lipid-like compounds is composed of nondegradable amino alcohols consisting of polar amine-containing head groups and nonpolar hydrocarbon tails [33,34]. A potent lipidoid C12 -- 200, was identified [34]. Mice received a single intravenous injection of C12 -- 200-formulated siRNA that targets serum FVII at either 0.1 or 1 mg/kg [34]. At both doses, complete silencing was observed at 24 h and protein levels returned to baseline within 20 days and 35 days for the 0.1 and 1 mg/kg doses, respectively. Additionally, C12 -- 200-formulated siTTR was administered to nonhumans [34]; C12 -- 200-formulated siTTR induced TTR silencing at 0.03 mg/kg [34], which is similar to the siTTR dose used in the MC3-SNALP formulation [30]. The C12 -- 200 lipidoid particle was generally well-tolerated in nonhumans at the dose levels tested. No significant changes in clinical chemistry parameters were observed except for a slight reduction in platelets, as well as a slight increase in ALT and AST levels. The C12 -- 200 lipidoid particle could also be used to deliver siRNA to the lungs via intranasal administration [34]. 3.2.3

Liposome-polycation-hyaluronic acid formulation

3.2.4

Both the SNALPs and the lipidoid particles demonstrated high in vivo efficacy. However, they have not been used as ‘targeting’ nanoparticles. The LPH formulation has been used to deliver siRNAs and miRs [35,36]. Cationic liposomes, composed of DOTAP and cholesterol, were prepared by thin film hydration method, followed by extrusion to form


5



small unilamellar vesicles. Protamine was added to a mixture of nucleic acids and hyaluronic acid, and then added to the small cationic vesicles, to form LPH. Intravenous injections of tumor-targeting LPH have demonstrated success in preclinical models [36]. LPH was mixed with PEG containing a malemide functional group, which allows single-chain variable antibody fragments to be conjugated to the LPH nanoparticles. GC4-targeted LPH was able to deliver siRNAs and miRs into mice bearing B16F10 lung metastasis, resulting in reduced lung metastasis and prolonged mice survival [36]. The levels of the proinflammatory cytokines (IL-6, IL-12 and IFN-g) and those of AST and ALT were very similar between animals injected with nucleic acids incorporated in the GC4-targeted LPH and those of untreated animals. Interestingly, nucleic acids formulated in the LPH formulation without the PEG outer shell induced a significant production of IL-6, IL-12 and IFN-g [36]. Therefore, the PEG outer layer in LPH is crucial in evading immune responses, similar to that in SNALPs and lipidoid particles. PEG has very low immunogenicity and is considered nontoxic. However, repeated injections of PEG-conjugated proteins and PEG-coated nanoparticles could elicit immune responses, including the activation of the complement system and the production of anti-PEG IgM antibodies, which could impair the safety of PEG-coated nanoparticles and the therapeutic efficacy of their drug cargo [37-42]. Furthermore, antibodies against PEG have been found in 22 -- 25% of healthy blood donors, up from 0.2% two decades ago, which could be the consequence of the increasing use of PEG in cosmetics, therapeutics and processed food [43]. To ensure that PEG-coated lipid nanoparticles could be used safely in nucleic acid delivery, patients should be prescreened for anti-PEG antibodies, and monitored for anti-PEG antibodies production and complement activation during the course of their treatment. Research in alternative types of hydrophilic polymers and peptides such as poly(hydroxyethyl-L-asparagine) and XTEN are being developed and may represent an alternative to PEG coating [44,45].

Delivery of nucleic acids by neutral lipid nanoparticles

3.3

We have used a different approach to avoid the toxicity issues associated with the first generation of cationic lipids. We developed a safe and highly effective neutral lipid-based nanoparticle to deliver antisense oligos and siRNAs to tumors following intravenous administration [15,46-50]. We selected 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to form our neutral liposomes because DOPC is natural, nonimmunogenic and highly versatile. DOPC liposomes are also physiologically more stable than DOPE liposomes. Additionally our experience with incorporation of drug molecules into liposomes taught us that liposomes composed of phosphatidylcholine are highly efficient in transporting drug molecules 6

to target cells [51-56]. Nucleic acids are mixed with the DOPC lipid. The nonionic surfactant Tween 20 is added to the mixture to reduce liposome size and aggregation. Excess tertiary butanol is added to the nucleic acid/DOPC/Tween 20 mixture, which is then frozen and lyophilized. When ready to use, the lyophilizate is reconstituted with normal saline to form lipid nanoparticles. The neutral DOPC nanoparticle has been used successfully to deliver nucleic acids systemically with remarkable efficacy in various orthotopic cancer models, including leukemia, ovarian, pancreatic and breast [15,46-50]. The characteristics of the various lipid-based nanoparticles in nucleic acid delivery have been summarized in Table 2. The safety, pharmacokinetics and efficacy of one DOPCincorporated antisense oligo formulation, BP-100-1.01, is currently being studied in a Phase I clinical trial. 4.

BP-100-1.01

Growth factor receptor bound protein-2 BP-100-1.01 is a DOPC-incorporated antisense oligo formulation designed to inhibit the production of the adaptor protein growth factor receptor-bound protein-2 (Grb-2). Though the Grb-2 protein does not have enzymatic activity, it is essential to cancer cell signaling (Figure 1). Grb-2 contains one Src Homology 2 (SH2) domain flanked by two Src Homology 3 (SH3) domains [57]. Grb-2 utilizes its SH2 domain to bind to phosphorylated tyrosine residues on YXNX sequence motifs in activated receptor/nonreceptor tyrosine kinases [57-60]. This binding allows proteins, like EGFR [57,60,61], Shc [58], platelet-derived growth factor receptor [57,62], insulin-like growth factor receptor substrate-1 [63-66], Bcr-Abl [67], focal adhesion kinase [68], ErbB2 [65,69], phospholipase C-g [70], phosphatidylinositol-3 kinase [71], fibroblast GFR (FGFR) [72], hepatocyte GFR [73] and NLM-ALK [74] to transduce signals to the activation of Ras [57-62,67], which in turn activates extracellular signals regulated kinases (Erk) [75-78] and Akt [79-85] for cancer progression. Alternatively, Grb-2 utilizes its SH3 domains to regulate cell signaling by binding to proline-rich motifs found in proteins like son-of-sevenless [60,86], synapsin [87], dynamin [88], C3G [89], c-Cbl [90-92]; 5-lipoxygenase [93], phosphatidylinositol-3 kinase [94,95], Grb-2-associated binder-1 [96], SLP-76 [97], protein kinase A [98], Vav [99] and NLM-ALK [74]. Thus, Grb2 has a critical role in cancer cell signaling, and inhibition of Grb-2 production has therapeutic potential. 4.1

Preclinical studies of BP-100-1.01 BP-100-1.01 targets the translation initiation site of the grb-2 transcript. The Grb-2 antisense oligo, composed of uncharged, nuclease-resistant P-ethoxy oligos, is incorporated into DOPC neutral nanoparticles [46,100-103]. In vitro studies demonstrated that BP-100-1.01 was effective in inhibiting the proliferation of Bcr-Abl-positive leukemic cell lines [100] as well as breast cancer cell lines that overexpress EGFR or ErbB2 leading to decreased Erk or Akt activation [101,102]. 4.2



Table 2. Comparison of the different lipid-mediated nucleic acid delivery strategies. Lipid system

Natural components

Potential toxicity or immunogenicity Efficacy Limitation Clinical status

SNALP Lipidoidz LPH§

Cholesterol, phosphatidylcholine Cholesterol Cholesterol, protamine, hyaluronic acid Neutral nanoparticle DOPC

Ionizable cationic lipids, PEG lipid-like molecules, PEG DOTAP, PEG

Lysosomal escape* Exocytosis* Lysosomal escape

Phase I Preclinical Preclinical

No

Lysosomal escape

Phase I

*Please refer to Section 6. z Lipidoid has potential utility as an intranasal delivery vehicle. § LPH contains a tissue-targeting moiety.


Grb-2 signaling

PY

Y P GRB-2

PY

YP Activates RAF

SOS

RAS GTP

Activates MEK Activates ERK

GDP Activates PI3K Activates AKT

Activates transcription

Figure 1. Grb-2 signaling. Grb-2 is recruited by activated tyrosine kinases to the activation of Erk and Akt. Erk: Extracellular signals regulated kinases; GDP: Guanine nucleotide diphosphate; Grb-2: Growth factor receptor bound protein-2; GTP: Guanine nucleotide triphosphate; MEK: Mitogen-activated protein kinase kinase; SOS: Son of sevenless.

BP-100-1.01 was also effective in inhibiting FGF-induced motility of breast cancer cells [103]. Pharmacology studies revealed that BP-100-1.01 exhibited a two-compartment model of distribution with a biphasic plasma clearance; t½a and t½b at ~ 6 min and 4 h, respectively [46]. Tissue distribution showed that BP-100-1.01 was widely distributed throughout the body. The tissue half-life of BP-100-1.01 was between 2 and 3 days [46]. Both mice and rabbits tolerated intravenous injections of BP-100-1.01very well. Their renal functions were evaluated by creatinine and blood urea nitrogen assays, while their hepatic functions were evaluated by serum glutamic-oxaloacetic transaminase and alkaline phosphatase assays. Mice were injected with 15 or 25 mg BP-100-1.01 per kg of body weight once a day for 5 consecutive days [46]. The renal and hepatic biochemical functions of mice injected with an accumulative dose of 125 mg of BP-100-1.01 per kg of body weight were not different from those of noninjected mice [46]. Rabbits were administered with eight intravenous injections of BP-100-1.01 (two injections per week for 4 consecutive weeks). The blood clotting time or the complement activation time of injected rabbits was not different from those of noninjected rabbits (data not shown). Intravenous injection of NOD/scid mice preconditioned by sub-lethal radiation with bcr-abl-positive leukemia cells

was demonstrated to cause a lethal leukemia syndrome involving marrow and spleen tissues; mouse survival varies between 15 and 25 days [104]. Using this animal model, we showed that BP-100-1.01, intravenously administered twice a week at 15 mg/kg body weight, increased the survival of mice bearing the bcr-abl-positive 32D leukemia xenografts; 80% of such mice had their survival extended to 32 -- 44 days [46]. The enhancement in mouse survival was specific to BP-100-1.01 as equivalent doses of DOPC nanoparticles or DOPC-incorporated control oligo did not increase mouse survival [46]. Since BP-100-1.01 induced survival benefit in a preclinical leukemia mouse model, and BP-100-1.01accumulated in the primary organs of leukemia manifestation: liver, spleen and bone marrow, we decided to evaluate the clinical activity of BP-100-1.01 in patients with leukemia. Preclinical studies of BP-100-1.01 were performed in gleevec-resistant leukemia cells because the clinical activity of BP-100-1.01 will be investigated in patients with refractory or relapsed leukemia. The gleevec-resistant K562R and BV173R leukemic cells, obtained from Dr. Nicholas Donato (University of Michigan, Ann Arbor, MI), were incubated with increasing concentrations of BP-100-1.01 or DOPCincorporated control oligo (Figure 2). BP-100-1.01 induced growth inhibition in the gleevec-resistant leukemic cells. The 50% growth inhibitory values of BP-100-1.01 in K562R and BV173R cells were ~ 12 and 9 µM, respectively (Figure 2). However, under identical conditions, DOPC-incorporated control oligo induced < 20% growth inhibition in the leukemic cells (Figure 2). Flow cytometry was used to analyze the percentages of BP-100-1.01-treated cells in the different phases of cell cycles. At 8 µM concentration, BP-100-1.01increased the percentage of sub-G1 cells from 2.8 to 16.5, and decreased the percentage of cells in G2/M phase from 20.9 to 10.9 (Table 3). These data indicate that BP-100-1.01predominantly induced growth inhibition and, to a lesser degree apoptosis, in gleevec-resistant leukemic cells. To confirm that BP-100-1.01 suppressed the expression of the Grb-2 protein target, BP-100-1.01 was administered to NOD/scid mice implanted with K562R tumors subcutaneously. Mice were given three intravenous injections of BP-100-1.01 or control DOPC liposomes. Compared to DOPC-treated tumors, BP-100-1.01-treated tumors had ~ 70% lower levels of Grb-2 protein, and ~ 90 -- 95%


7


BV173R cells


Cell viability (% of untreated cells)

K562R cells 120

120 100 80 60 40 20 0

100 80 60 40 20 0 0

3

6

9

12 15 0 3 Liposomal oligos (mm)

6

9

12

Figure 2. BP-100-1.01 induced growth inhibition in gleevec-resistant leukemia cells. Gleevec-resistant K562R and BV173R cells were incubated with 0 -- 12 µM of BP-100-1.01 (&) or DOPC-incorporated control oligo (~) for 4 days. The alamarBlue dye incorporation method was used to determine the growth of the leukemic cells. Growth of treated cells was compared to that of untreated cells, and expressed as percentage of untreated cells. DOPC: Dioleoyl-sn-glycero-3-phosphatidylcholine.

Table 3. BP-100-1.01 induced apoptosis in gleevec-resistant leukemia cells. BP-100-1.01* (mM)

sub-G1 (%)

G1 (%)

S (%)

G2/M (%)

0 4 8

2.8 ± 0.3 9.7 ± 0.6 16.5 ± 1.4

50.3 ± 1.4 54.6 ± 0.6 50.0 ± 1.5

26.1 ± 1.1 23.0 ± 0.4 22.8 ± 0.6

20.9 ± 1.7 13.0 ± 0.4 10.9 ± 0.2

*BV173R leukemic cells were incubated with BP-100-1.01 for 4 days and then processed for propidium iodide staining. Percentages of cells in the different phases of cell cycle were analyzed by flow cytometry.

lower levels of Grb-2 downstream partners, phosphorylated Erk and phosphorylated Akt (Figure 3). Based on the results of these preclinical studies, a Phase I clinical trial of BP-100-1.01 was initiated in patients with refractory or relapsed hematologic malignancies.

Phase I clinical trial of BP-100-1.01 The objective of the Phase I clinical trial of BP-100-1.01 was to define the safety, maximum tolerated dose (MTD), optimal biologically active dose, pharmacokinetics and antileukemia activity of BP-100-1.01 in patients with relapsed or refractory hematologic malignancies. The study is a standard 3 + 3 Phase I dose-finding study in patients with relapsed or refractory chronic myeloid leukemia in blast phase (CMLBP), acute myeloid leukemia, acute lymphoblastic leukemia and myeloid dysplastic syndrome. As MTD was not observed in the preclinical studies, the starting clinical trial dose was based on the predicted efficacy dose, which was 45 mg/m2; the starting dose of BP-100-1.01 was 1/10 of the predicted efficacy dose, that is, 5 mg/m2. Based on the preclinical tissue distribution studies, BP-100-1.01 is administered to patients twice weekly. The drug is given intravenously over 2 -- 3 h for 28 days. Dose escalation has proceeded through 10, 20, 40 and 60 mg/m2. In a preliminary report, a total of 28 patients was included in cohorts treated at a dose of 5 mg/m2 in Cohort 1 (n = 13), 10 mg/m2 in Cohort 2 (n = 6), 20 mg/m2 in Cohorts 4.3

8

3 (n = 3), 40 mg/m2 in Cohort 4 (n = 3) and 60 mg/m2 in Cohort 5 (n = 3). One patient was reported to experience a dose-limiting toxicity (DLT) of grade 3 mucositis and handfoot syndrome, while receiving concurrent hydroxyurea for proliferative CML-BP treated in Cohort 1 (dose 5 mg/m2). No other BP-100-1.01-related toxicity has been noted in any of the subsequent patients treated at this or subsequent dose levels, with no DLT and no MTD identified to the time of this preliminary report. Nine of the 18 evaluable patients treated were reported to have at least a 50% reduction in peripheral or bone marrow blasts from baseline. Flow cytometric analysis was performed on peripheral blood samples collected from patients in Cohorts 3, 4 and 5, which correspond to 20, 40 and 60 mg/m2 of BP-100-1.01. Blood samples were collected prior to study initiation and during therapy. A significant (i.e., ‡ 50%) decrease in the levels of Grb-2 protein and its downstream signaling partner, phosphorylated Erk, was found in at least half of the patients. Enrollment and dose escalation continue in this trial. 5.

Conclusion

Targeting oncogenic proteins has been pursued as a means of specific therapy in cancer for many years as a means of delivering more directed and less toxic therapy. One approach to do this has been the disruption of the translation of such proteins through nucleic acids. Issues with serum stability and cell penetration have hindered the ability to provide effective


DOPC control

BP-100-1.01


BP-100-1.01 decreased Grb-2 protein

Grb-2 protein expression by 70%

β-actin Phosphorylated Erk

Phosphorylated Erk by 90%

β-actin


Phosphorylated Akt

Phosphorylated Akt by 95%

β-actin

Figure 3. BP-100-1.01 decreased the expression of Grb-2 protein and its downstream partners in vivo. K562R cells were implanted subcutaneously in NOD/SCID mice. When the tumors were ~ 0.5 cm in diameter, mice were injected intravenously with 15 mg of BP-100-1.01 per kg of mouse body weight or an equivalent dose of control DOPC liposomes on days 1, 4 and 7. Mice were sacrificed on day 9, and tumors were processed. Western blots were performed to determine the tumor expression of Grb-2 protein, and its downstream signaling partners phosphorylated Erk and phosphorylated Akt. DOPC: Dioleoyl-sn-glycero-3-phosphatidylcholine; Erk: Extracellular signals regulated kinases.

therapy with these approaches. Significant progress has been made in our understanding of these issues and has resulted in development of more effective means of delivering such therapies. BP-100-1.01 is a specific Grb-2 antisense oligo incorporated into neutral liposomes. Grb-2 is an attractive target because, despite not having specific enzymatic activity of its own, it serves as a linker to transduce signals form a number of oncogenic proteins that result in the activation of Ras. Early results of a Phase I trial in leukemia suggest that BP-100-1.01 effectively reduces Grb-2 protein expression, resulting in inhibition of downstream effectors such as phosphorylated Erk at doses that have not resulted in DLT. Studies with BP-100-1.01 are continuing and expanding to other indications as well as combinations to fully understand the role BP-100-1.01 may have in cancer therapy. 6.

Expert opinion

Earlier work in the nucleic acid field was devoted to engineering nucleic acids with enhanced nuclease stability. Recent work is devoted to developing synthetic or natural nanoparticles that will enable the systemic delivery of nucleic acids to disease sites. In this article, we focus on lipid-based nanoparticles because they are some of the most advanced in vivo carriers to date, and are being used in clinical trials. Efficient target knockdown by antisense oligo or siRNA has been demonstrated in rodents, primates and recently in humans. Nonetheless, more work is needed to realize the clinical potential of lipid-mediated delivery of nucleic acids. There is much success in the delivery of nucleic acids by lipid nanoparticles to tumors that have leaky blood vessels and large

fenestrations between endothelial cells. However, lipid nanoparticles do not readily diffuse throughout the interstitial space of a tumor. To gain access into tumors with poor vascularization, lipid nanoparticles with different properties will have to be developed. We believe that the design of such nanoparticles will be aided by using three-dimensional cultures, or monolayer cultures that are layered on top of extracellular matrices. This is because monolayer cultures lack many of the characteristics of those found in tumors, including a nutritional gradient and a microenvironment that affect cellular signaling, which in turn influences cellular interactions with lipid nanoparticles. Multicellular tumor spheroids of size 200 -- 500 µm have been reported to be better predictors of in vivo drug activity than monolayer cultures. Carver et al. [105] observed differences in the uptake and activity of lipid nanoparticles between spheroids and monolayer cultures. The CNS is likely the most challenging delivery site for lipid nanoparticles, as the lipid nanoparticles have to cross the blood--brain barrier, which is lined with tight junctions. Currently drug delivery across the blood--brain barrier has focused on directly bypassing the blood--brain barrier using convection enhanced delivery [106] or intrathecal injections [107]. In a recent Phase II clinical trial, convection-enhanced delivery was used to infuse patients’ brain tumors with the trabedersen (AP 12009) antisense oligo, which is a phosphorothioate oligo that targets the human TGF-b2 gene [106]. Trabedersen was administered at two different doses -- a low dose of 2.48 mg (10 µM) and a high dose of 19.81 mg (80 µM). The frequency of patients with drug-related adverse events was higher in the 80 µM-trabedersen group (43%) than in the 10 µM-trabedersen group (27%), suggesting that


9



a lower dose of phosphorothioate antisense oligos should be used in order to avoid phosphorothioate toxicity in the CNS. Unfortunately, the trabedersen Phase III clinical trial was terminated early as preliminary data analysis revealed unfavorable risk-to-benefit effect was observed with the 10-µM drug treatment. In a recent Phase I clinical trial, ISIS 333611, an antisense oligo that reduces expression of wild-type and mutant human superoxide dismutase 1 protein [107], was given to patients as an intrathecal infusion. ISIS 333611 (ranging between 0.15 and 3 mg) or placebo was infused intrathecally for over 11 h. The most common adverse events were post-lumbar puncture syndrome and back pain, which did not differ between ISIS 333611 and placebo groups. ISIS 333611 was detected in patients’ cerebrospinal fluid; however, it is not known whether the drug was able to exit from the cerebrospinal fluid and enter into the brain parenchyma. Since nucleic acids are large in molecular weight (ranging between 6 and 14 kDa) and are not expected to cross plasma membranes and penetrate into brain cells, it is highly probable that nanoparticles are needed to assist nucleic acid internalization into the brain parenchyma even when the nucleic acids are delivered intrathecally. Lipid nanoparticles are internalized by cells via endocytosis; the predominant mechanism being macro-pinocytosis. The MC3-SNALP nanoparticle is one of the most efficient lipid nanoparticle in siRNA delivery. Yet surprisingly, Gilleron et al. [108] reported that < 2% of the internalized siRNA cargo delivered by the MC3-SNALP was able to escape from the endocytic system and enter the cytoplasm. There are reports that a minor pathway, probably mediated by fusion between cationic lipid nanoparticles and the plasma membrane, is responsible for the trafficking of siRNA to the cytoplasm [109,110]. It is crucial to identify the uptake and trafficking mechanisms of nucleic acid drugs incorporated in the different lipid nanoparticle formulations so that superior lipid Bibliography

nanoparticles could be engineered. Additionally, these studies may enable the utility of small molecules, like Retro-1, in enhancing the activity of nucleic acid drugs by increasing their accumulation in the cytoplasm [111]. Sahay et al. [112] demonstrated that the C12-200 lipidoid nanoparticle bypassed early endosomes and delivered the siRNA cargo directly to late endosomes, where ~ 70% of the internalized cargo was exocytosed to the external milieu. The transmembrane glycoprotein Niemann-Pick type C1 (NPC1) was critical in regulating siRNA exocytosis as late endosomes and lysosomes from NPC1-negative mouse embryonic fibroblasts were found to retain ~ 15-fold higher siRNA levels than their wild-type counterparts [112]. These studies may have uncovered a resistance mechanism of nucleic acid drugs that was not known before. Resistance to nucleic acids could be a major barrier in the clinical application of nucleic acidbased therapeutic, particularly in chronic diseases where multiple or life-long nucleic acids administration is likely. Future studies are needed to improve our understanding of how different lipid nanoparticle design may invoke diverse cargo release mechanisms and/or nucleic acid resistance mechanisms. Understanding these mechanisms should further our ability in designing superior lipid nanoparticles and in translating the potential of nucleic acid cancer drugs to the clinic.

Declaration of interest AT Ashizawa is the Director of Preclinical Operations and Research at BioPath Holdings, Inc. J Cortes has received research support from BioPath Holdings, Inc. The authors have no other 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 apart from those disclosed.

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Affiliation

Ana Tari Ashizawa*1,2 & Jorge Cortes†3 † ,*Authors for correspondence 1 BioPath Holdings, Inc., 4710 Bellaire Blvd Suite 210, Houston, TX 77401, USA Tel: +1 713 385 4392; E-mail: [email protected] 2 University of Florida, The Department of Neuroscience, Gainesville, FL, USA 3 University of Texas MD Anderson Cancer Center, Department of Leukemia, 1515 Holcombe Blvd., Unit 428, Houston, TX 77030, USA. Tel: +1 713 794 5783; Fax: +1 713 794 4297; E-mail: [email protected]

Stimuli-responsive liposomes for the delivery of nucleic acid therapeutics.

Enhanced anticancer efficacy by ATP-mediated liposomal drug delivery.

Precision spherical nucleic acids for delivery of anticancer drugs.

Liposomal spherical nucleic acids.

Structurally engineered anodic alumina nanotubes as nano-carriers for delivery of anticancer therapeutics.

Development of synthetic lethality anticancer therapeutics.

Targeting Lipid Metabolic Reprogramming as Anticancer Therapeutics.

Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics.

Clinically relevant anticancer polymer Paclitaxel therapeutics.

Layer-by-layer assembly of liposomal nanoparticles with PEGylated polyelectrolytes enhances systemic delivery of multiple anticancer drugs.

Delivery materials for siRNA therapeutics.

New Developments in Liposomal Drug Delivery.

Preclinical and Clinical Advances of GalNAc-Decorated Nucleic Acid Therapeutics.

Nucleic acid aptamers: research tools in disease diagnostics and therapeutics.

Agile delivery of protein therapeutics to CNS.

Delivery of cancer therapeutics using nanotechnology.

Long-term delivery of protein therapeutics.

Non-viral nucleic acid containing nanoparticles as cancer therapeutics.

Nanodelivery systems for nucleic acid therapeutics in drug resistant tumors.

Intracellular trafficking of new anticancer therapeutics: antibody-drug conjugates.

Prospects for nucleic acid-based therapeutics against hepatitis C virus.

Cellular delivery of peptide nucleic acids (PNAs).

Electroporation-enhanced delivery of nucleic acid vaccines.

Cells as delivery vehicles for cancer therapeutics.