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Why DNA?

Genisphere is the only commercial source of drug delivery nanocarriers made entirely out of DNA. Using DNA as a biomaterial for manufacturing offers several inherent advantages including scalability, stability, and precise control of the final size and shape of 3DNA. The multivalency of 3DNA allows finely tuned targeting capabilities and high capacity for a variety of payloads. The modular and flexible properties of 3DNA make it easy for Genisphere to quickly develop biotherapeutics for a variety of indications.

3DNA Nanostructure

3DNA is Genisphere's core nanotechnology: a 3-dimensional structure made entirely out of DNA. The 3-dimensional structure is a highly branched molecule built from interconnected monomeric subunits of DNA.

The 3DNA Monomer

3DNA Monomer
3DNA Monomer

A 3DNA monomer is composed of two DNA strands that share a central region of sequence complementarity. When the two strands anneal, a monomer is formed, with a central double-stranded 'waist' bordered by four single-stranded 'arms'. In most cases all of the nucleic acids in the DNA strands are natural; however, modified DNA bases may also be used. The branched structure of a 3DNA monomer is simply due to complementary base pairing in the central region, and lack of base pairing in the peripheral regions. Monomers are chemically cross-linked to prevent strand separation. Typically, Genisphere uses seven different strands to prepare five unique monomers. Each monomer has two terminal 5' ends and two terminal 3' ends. By design, the peripheral sequence of both 5' ends and both 3' ends are either the same or different DNA sequence, depending on the monomer.

The Layered Architecture of 3DNA

The single-stranded arms of each of five unique monomers base-pair with one another according to their specific sequences. Base-pairing between the arms of complementary monomers allows directed assembly of the 3DNA as a step-wise process of forming layers. Construction of a 3DNA structure begins with a single initiator monomer, to which a first layer of monomers is hybridized. The result is a one-layer 3DNA configuration with 12 single-strand arms on the outer surface. This assembly is chemically cross-linked to prevent dissociation. Next, a second layer of monomers is attached to the outer arms of the first layer by the same hybridizing and crosslinking steps. In this two-layer 3DNA scaffold the number of free single-stranded arms increases to 36; half have terminal 5' ends and half have terminal 3' ends.

1 layer 3DNA
1-layer 3DNA
2 layer 3DNA
2-layer 3DNA

Characteristics of the 3DNA Core

Depending on the selection of DNA strands and monomers used during manufacturing, typical 2-layer 3DNA nanostructures have a diameter of 60nm, a molecular weight of 1000kD, and a Zetapotential of -30mV. The cross-linked regions delay dissociation of the DNA strands yet maintain overall flexibility and mobility of 3DNA in vitro and in vivo. The core 3DNA nanoscaffold is 99% solvent in most aqueous media due to its dynamic DNA configuration. 3DNA is biocompatible and has no demonstrated toxicity to living cells.

3DNA Platform

After the 3DNA core is manufactured it is further functionalized with targeting devices and labels or payload conjugates. These molecules can be hybridized to the single-stranded arms of the 3DNA nanoscaffold as oligonucleotides or as oligonucleotide conjugates. Some molecules can intercalate into the double-stranded core regions of the 3DNA nanostructure. The flexibility of the 3DNA platform to carry tracking labels to targeted tissues provides an option to incorporate a companion diagnostic into the drug development process. Example reagents used in targeted therapeutics are shown below: both are 2-layer 3DNA with four molecules of folic acid for targeting. Cy3 labels were attached to arms of 3DNA for a simple biodistribution study. Doxorubicin was intercalated into the core of 3DNA for a preliminary ovarian tumor efficacy study.

Folic Acid 3DNA Cy3
Folic Acid:3DNA:Cy3
Folic Acid 3DNA Dox
Folic Acid:3DNA:Doxorubicin

Targeting Conjugates

antibody oligo conjugate
Antibody-Oligo Conjugate

Cell-specific targeting may be achieved by specific antibodies, antibody fragments, peptides or aptamers. Various heterobifunctional crosslinkers can be used to conjugate proteins to short DNA oligos which hybridize with the peripheral arms of 3DNA. Site-specific conjugation strategies can also be used to ensure a 1:1 ratio of DNA to targeting protein. Dual or multiple targeting is fully enabled by the architecture of the 3DNA and may help reduce off-target effects. Unique conjugates can be separately titrated to the 3DNA nanocarrier to determine the ideal ratios by experimentation. The unique and distinct multivalent targeting powered by 3DNA can promote cellular uptake, alter pharmacokinetics, or change the mechanism of action.

antibody on 3DNA
Antibody Conjugates on 3DNA
two unique antibodies on 3DNA
Two Antibody Conjugates on 3DNA

ELISAs or other assays can be used to confirm bioconjugation chemistry does not alter the ability of the targeting molecule to bind its target. Specificity of targeting may also be tested by hybridizing the targeting conjugate to 3DNA cross-linked with fluorescent DNA oligos. Labels such as Cy3 are helpful to confirm targeting by simple cell staining or microscopy of tissue sections, while dyes including Alexa750 can be used for whole organ or whole animal imaging.

Payload Conjugates

3DNA cargo can consist of small drug molecules, peptides, or proteins. The release of payload from internal cellular compartments is facilitated by labile linkers or biological breakdown of DNA. The number of payload molecules depends on the design of the DNA oligo to which the small molecule is conjugated. When the DNA oligo contains multiple conjugation sites, and several multi-drug conjugates hybridize to 3DNA, the overall amount of payload delivered is increased.

oligo with three drug molecules
Oligo with 3 Drug Molecules
18 drug molecules on 3DNA
18 Drug Molecules on 3DNA
18 drug molecules on 3DNA with 4 targeting antibodies
18 Drug Molecules on 3DNA with 4 Targeting Antibodies

targeted nucleic acids
Targeted Nucleic Acid

Nucleic acids have also been successfully delivered with 3DNA. Plasmids can be cut with a restriction enzyme and ligated with a short DNA sequence complementary to 3DNA. Double-stranded siRNA or miRNA can be designed to contain bases with improved stability in vivo, and a short extension of DNA which hybridizes 3DNA. 3DNA formulations have demonstrated efficacy in animal models delivering siRNA to multiple targets outside the liver, including pancreatic and ovarian cancers. 3DNA:miRNA treatment demonstrated blood brain barrier penetration and accumulation in microglia, a unique immune cell population in the brain.