Auto-recognizing therapeutic RNA/DNA chimeric nanoparticles (NP)
Inventors
Shapiro, Bruce A. • Afonin, Kirill A. • Viard, Mathias D. • Bindewald, Eckart H. • Jaeger, Luc • Santhanam, Arti N.
Assignees
University of California San Diego UCSD • US Department of Health and Human Services
Publication Number
US-9631192-B2
Publication Date
2017-04-25
Expiration Date
2032-11-19
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Abstract
Auto-recognizing therapeutic R/DNA chimeric nanoparticles (R/DNA NP) are described that are pairs of DNA/RNA hybrids where the DNA molecules have complementary toehold sequences that promote the re-association of the R/DNA NPs when mixed resulting in the formation of RNA/RNA duplexes that act as siRNAs.
Core Innovation
The invention provides RNA/DNA chimeric polyfunctional nanoparticles (R/DNA NP) consisting of at least two chimeric nanoparticles where the DNA molecules contain complementary toehold sequences. This design promotes re-association of the R/DNA nanoparticles when mixed, resulting in the formation of RNA/RNA duplexes that operate as siRNAs. These siRNAs can inhibit target RNAs associated with disease processes, including apoptosis inhibitor proteins and pathogenic agents such as HIV.
The problem addressed by the invention lies in the challenges associated with clinical delivery of RNA interference (RNAi) therapies. Conventional exogenous siRNAs suffer from intravascular degradation, limited tissue specificity, and difficulties in tracking delivery and response in vivo. The invention overcomes these obstacles by splitting siRNA duplexes into two inactive RNA/DNA hybrids that recognize and re-associate inside target cells via complementary toehold sequences, forming active siRNAs for gene silencing.
The invention further enables improved pharmacodynamics by allowing the incorporation of various chemical modifications and functional moieties on DNA strands without interfering with siRNA activity. These functional moieties may include fluorescent tags for imaging, recognition domains for targeting diseased cells, and other functionalities that enhance delivery, specificity, and therapeutic effect. The nanoparticle design permits multiple functionalities to be introduced, expanding the therapeutic and diagnostic capabilities.
Claims Coverage
The patent claims include one independent claim focusing on the composition of R/DNA chimeric polyfunctional nanoparticles with complementary toehold sequences and their functional re-association, along with methods of use. The claims disclose multiple inventive features related to the nanoparticle structure, functionalities, and therapeutic applications.
Composition of complementary auto-recognizing RNA/DNA chimeric nanoparticles
The invention features an R/DNA chimeric polyfunctional nanoparticle comprising at least two complementary auto-recognizing chimeric nanoparticles. Each chimeric nanoparticle consists of a DNA oligonucleotide hybridized to one or more complementary RNA oligonucleotides. The DNA oligonucleotides have complementary 5′ and 3′ toehold sequences that hybridize and promote reassociation, resulting in a DNA duplex and one or more functional RNA duplexes.
Formation of functional siRNA duplexes through DNA toehold-mediated reassociation
The complementary toehold sequences in the DNA strands drive the reassociation of the two chimeric nanoparticles, which releases functional RNA duplexes that serve as Dicer substrates. These Dicer substrates can be converted by dicing into siRNA molecules that inhibit target RNAs.
Targeting therapeutically relevant RNAs including apoptosis inhibitor proteins and pathogenic genomes
The siRNAs produced upon reassociation can inhibit RNAs that provide therapeutic benefit when silenced. This includes RNAs encoding proteins involved in disease processes such as apoptosis inhibitor proteins (including BCL-2, FLIP, STAT3, XIAP) and pathogenic RNA genomes like those from HIV-1.
Attachment of functional moieties for targeting and imaging
The chimeric nanoparticles can include functional moieties such as recognition domains and fluorescent tags attached to the DNA strands. These moieties facilitate specific binding to target cells (e.g., diseased or infected cells) and tracking of nanoparticle delivery and reassociation via fluorescence resonance energy transfer (FRET).
Pharmaceutical compositions and kits comprising the R/DNA nanoparticles
Claims also cover pharmaceutical compositions containing the R/DNA chimeric polyfunctional nanoparticles with pharmaceutically acceptable excipients, and kits containing these nanoparticles for therapeutic uses.
Methods of inhibiting gene expression in cells
Methods are claimed for inhibiting the expression of HIV-1 or cancer genes in cells by contacting the cells with effective amounts of the R/DNA chimeric polyfunctional nanoparticles that release siRNAs upon dicing.
Generation of multiple functionalities and siRNA duplexes from reassociation
The nanoparticles can be designed to generate multiple functional RNA duplexes, including multiple Dicer substrates capable of forming siRNAs, which may target the same or different target RNAs. Additional functionalities such as RNA aptamers (e.g., Malachite Green RNA aptamer producing fluorescence upon duplex formation) can also be incorporated.
Complementary toehold sequences at both ends of the chimeric nanoparticles
The design can include complementary 5′ and 3′ toehold sequences at both ends of the first and second DNA oligonucleotides to provide extended complementary toeholds that enhance reassociation.
The claims of the patent comprehensively cover the composition of RNA/DNA chimeric polyfunctional nanoparticles featuring complementary toehold-mediated reassociation leading to siRNA release, the incorporation of functional moieties for targeting and imaging, pharmaceutical formulations and kits including these nanoparticles, and therapeutic methods to inhibit HIV-1 and cancer gene expression. The claims also encompass the generation of multiple functionalities and use of complementary toeholds at both DNA strand ends to optimize nanoparticle reassociation.
Stated Advantages
Reduced intravascular degradation due to increased stability of RNA/DNA hybrids compared to siRNAs.
Enhanced tissue specificity enabled by chemical modifications on DNA strands allowing targeted delivery to diseased cells.
Improved pharmacodynamics through fluorescent tagging allowing real-time imaging of delivery and response using FRET.
Capability to introduce multiple functionalities including targeting moieties, imaging tags, and therapeutic siRNAs without interfering with RNAi processivity.
Prolonged effect of gene silencing compared to pre-formed siRNAs.
Higher control over targeting specificity and potential for controlled therapeutic activation inside target cells.
Documented Applications
Therapeutic silencing of target genes in cells, with specific examples including inhibiting HIV-1 genes and cancer-related genes such as apoptosis inhibitors.
Use in treatment of viral infections including HIV by targeting infected cells to induce apoptosis through combinatorial RNA interference.
Targeted gene silencing in neoplastic cells leading to treatment of neoplasias or cancers such as breast cancer, prostate cancer, melanoma, glioblastomas, colon cancer, ovarian cancer, and non-small cell lung cancer.
In vivo delivery and imaging of RNAi therapeutics with enhanced tumor uptake demonstrated in mouse xenograft models.
Combination therapies with second therapeutic agents such as anti-viral or anti-cancer agents.
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