targeted delivery have emerged as unique strategies for improving therapeutic
efficacy while minimizing toxicity and off-target effects. Targeted
nanocarriers can be used to deliver novel drug candidates and to provide new
formulations of matured therapeutics. Multivalent-engineered nanostructures also
hold the possibility to deliver combination therapeutics.1-3 There
are several nanotech-based drugs already launched and hundreds of new targeted
treatments under development.
joins the list of targeted drug delivery nanoreagents, including by broad
definition, liposomes, quantum dots, gold nanoshells, micelles, magnetic
nanoparticles, dendrimers, and carbon nanotubes. While a variety of polymeric
materials are used to manufacture these nanocarriers, Genisphere’s 3DNA nanotechnology
platform is DNA-based and is composed of a cross-linked network of uniquely
designed DNA strands.4 Looking to expertise in both Pharma and
Academia, Genisphere has been leveraging a collaborative model to advance
several drug delivery programs, and is also progressing its own lead compounds
based on the 3DNA platform.
manufactures therapeutic quantities of drug delivery nanocarriers called 3DNA.
The manufacturing process is intended to consider the need for nanocarriers
with a truly modular design. Like other nanoreagents, 3DNA is constructed using
materials, in this case, DNA, that are not new to the FDA.
uses seven unique sequences of single-stranded DNA designed to hybridize to one
another to create building block monomers, each with a central double-stranded region
and four terminal single-stranded regions (Figure 1). By design, it is possible
to manufacture five unique monomers. The monomers themselves hybridize to each other
in specific orientations, due to base-pairing between complementary single-stranded
regions. For assembly, monomers are hybridized and crosslinked to each other in
a step-wise fashion of forming layers (Figure 1), and a two-layer core 3DNA
nanoscaffold with a diameter of 60 nm is typically used for drug delivery
applications. Importantly, crosslinking during manufacturing ensures the
stability of the core 3DNA structure, providing a shield from the harsh physiological
environments often found in vivo.
functionalized 3DNA with a variety of targeting devices and drug cargoes,
depending on the intended use. Therapeutics successfully conjugated to 3DNA
include small molecules, proteins, peptides, miRNA, siRNA, mRNA, and plasmid DNA.
Typical targeting devices are selected based on an up-regulated marker on the cell
of interest, and include antibodies, antibody fragments, peptides, sugars,
vitamins, and other targeting devices. Like other nanocarriers, 3DNA uses
targeting devices to ensure specificity and minimize off-target drug effects.
Similarly, sustained-release formulations are possible to manufacture, by modulating
attachment chemistry, using PEG or other stealth molecules, or incorporating
into a hydrogel formulation.
DNA as a nanotechnology
matrix offers a unique set of physical, chemical, and biological properties.5,6
Because 3DNA is completely and only made of DNA, it is soluble, biocompatible,
and biodegradable into non-toxic material, with a metabolic clearance pathway
already known to the host organism. Given its unique properties, structural
flexibility and solubility, 3DNA is not really a nanoparticle, but rather a
nanocarrier. Using DNA offers versatility in assembly and control of the
physical size or shape of the nanocarrier. 3DNA is uniformly negatively
charged, and is designed with a base composition to avoid activation of the immune
response typically characterized by cytokine production and up-regulation. Biodistribution,
accumulation at desired site, and clearance are only determined by 3DNA targeting
moieties, not by the core structure.
3DNA has hundreds of drug
attachment sites that can allow lower doses to achieve the same efficacy as
free drug when the appropriate targeting moiety is selected. In addition, 3DNA
is completely customizable for targeting or multi-targeting with two or more
unique moieties per single nanocarrier. This flexibility in multivalency
enables delivery of a targeted combination therapeutic to, for example, engage
the immune system while delivering a cytotoxin to disease cells. Because there
are many options for conjugating DNA to drugs, 3DNA enables delivery of a broad
range of drug compounds. Multiple linker chemistries can be used in the same
construct, enabling differential drug release. Importantly, two or more
different drugs may be coupled to the same 3DNA molecule, in both the core and
also on the periphery, further maximizing the therapeutic payload.
In mouse and rabbit
models, no toxicity has been observed at therapeutic dose levels or after
chronic treatment of various 3DNA formulations. Throughout the course of these
studies, all animals appeared healthy, demonstrated normal behavior, and showed
no signs of toxic effects nor change in body weight due to the administration of
the 3DNA nanocarrier. Immunocompetent mice demonstrated little to no cytokine
activation 0.5-48 hours after systemic administration of 3DNA nanocarriers.
Both deep sequencing and PCR have been used to show 3DNA nanoscaffolds are degraded
after cargo delivery in vivo, with no impact/integration to target cell genome.
To study biodistribution
using radiolabeled antibodies, mice were injected with either radiolabelled
anti-ICAM antibody, or a 3DNA targeted against the same radiolabeled anti-ICAM
antibody. The animals were sacrificed, organs were excised, and isotope was
counted and compared to determine the presence of control antibody versus targeted
3DNA. Interestingly, only the liver showed similar distribution between the two
delivery systems, while kidney, brain, spleen, heart, and lung showed higher
presence of 3DNA, suggesting 3DNA did not accumulate in the liver like other
nanoparticles. Of particular note was the 3DNA crossing the blood-brain barrier
In another simple
biodistribution study (Figure 2), folic acid-targeted, Cy3-labeled 3DNA was
compared to untargeted Cy3-labed 3DNA (no drug cargo was used). Various organs
were collected immediately prior to sacrificing the mice at specific time
points post injection. Samples from non-injected animals showed minimal levels
of auto-fluorescence and no nanostructure-like fluorescence, as expected.
Untargeted 3DNA nanocarriers did not accumulate in tissues, while targeted 3DNA
nanocarriers accumulated only in tissues expressing the folate receptor.
Specifically, 3DNA nanocarriers with folic acid attached as the targeting
molecule bound to specific cells in the brain, liver, and lung within 4-24
hours following injection.
Targeted Delivery of Payload
Genisphere pursued a
collaboration with Dr. Janet Sawicki at the Lankenau Institute for Medical
Research to demonstrate functional in vivo delivery of a cargo molecule. Specifically,
commercially available luciferase mRNA was combined with a specially designed
3DNA containing folate as a targeting molecule (FA-3DNAmRNA). The 3DNA was
designed to bind the mRNA via a sequence within the mRNA itself. As a control, untargeted
3DNA with luciferase mRNA (3DNAmRNA) was also prepared. Each of the
formulations as well as vehicle were intraperitoneally injected into mice
bearing orthotopic ovarian tumors. At various time points, the animals were
imaged for bioluminescence to determine the relative luciferase expression over
time (Figure 3). Based on the imaging results, the ovarian tumors were the only
observable targeted tissue, maximal mRNA expression occurred 6 hours after
injection, and targeting was required for efficient delivery. Untargeted 3DNA
demonstrated minimal luciferase expression, further supporting the requirement
for targeting and the overall lack of an EPR dominant effect.
APPLICATION IN OPHTHALMOLOGY
Cataract surgery is one of
the most common procedures performed throughout the world, but it is not
without complications. While surgery restores vision in the majority of cases,
up to 40% of adults and most children develop a secondary cataract, a
vision-impairing condition called posterior capsule opacification (PCO). Laser
treatment is required to correct PCO, but it is expensive and can be risky.
Genisphere's therapeutic candidate GL-249 has been designed as a quick and easy
treatment to prevent PCO at the time of cataract surgery, by immunodepleting the
cells that cause PCO.
The specific cells in the
eye that contract and produce wrinkles leading to PCO can be targeted by a
unique monoclonal antibody. This antibody is used as a targeting device on 3DNA
loaded with doxorubicin, a cytotoxic drug, and the resulting GL-249 formulation
has been studied in mouse and rabbit models. Rabbits aggressively develop PCO 4
weeks after cataract surgery, and in one study, GL-249 reduced the incidence of
the condition in rabbits compared to controls when delivered at the time of
cataract surgery.7 Further development of sustained-release
formulations may lead to a quick and easy method to prevent PCO at the time of
cataract surgery, and open the door to treating other serious conditions of the
APPLICATION IN ONCOLOGY
In a preliminary study,
ovarian tumor-bearing mice were injected with untargeted Cy3-labeled 3DNA or
with Cy3-labeled 3DNA with either folic acid, anti-transferrin receptor
antibody, or antifolate receptor antibody-targeting moieties. The animals were
sacrificed, and various tissue sections were prepared and viewed to confirm the
expected biodistribution of targeted 3DNA. The targeted Cy3-3DNA was observed
in the center area of the tumor, with little staining in the stroma, while
untargeted Cy3-labelled 3DNA did not localize to the tumor. To assess if the various
targeted Cy3-labeled 3DNA were directed to the tumor via macrophage uptake, ovarian
tumor sections were labeled with F4/80 antibody to detect macrophages.
Macrophage staining was observed in cells in the stroma and adipose tissue (when
present), and a few single cells in the tumor demonstrated little co-localization
with the Cy3-labeled (3DNA targeted) tumor cells. Because no dual-labeling was
observed, it is believed the presence of targeted, Cy3-labeled 3DNA observed in
the tumors is not due to macrophage uptake and is a result of true targeting.
To test delivery of a
small drug molecule, mice bearing ovarian tumors derived from ID8-Fluc
bioluminescing cells were systemically injected twice a week for 3 weeks. The
injected formulations included the cytotoxin doxorubicin, delivered as free
drug or delivered using folate-targeted 3DNA. At the end of the short study, it
was observed doxorubicin delivered with folate-targeted 3DNA significantly
hindered ovarian tumor growth compared to the same dose of free doxorubicin
A similar mouse ovarian
tumor model was used in a study using folate-targeted 3DNA formulations to
deliver siRNA (lead candidate GL-233).8 Human antigen R (HuR), also
called ELAVL1, is an RNA-binding protein that regulates the expression of genes
known to function in tumor cell survival and in drug resistance. A double-stranded
siRNA to HuR was designed with modified RNA bases for stability, and an extension
of 23 ribonucleotides on the 3’ end of the passenger strand for hybridization to
single-stranded peripheral portions of 3DNA. In this case, mice bearing ovarian
tumors derived from ID8-Fluc bioluminescing cells were intraperitoneally injected
twice a week for 4 weeks. Both tumor growth and ascites development were
reduced in animals treated with 3DNA reagents compared to control formulations.
A follow-up study (Figure 5) of the folate-targeted siHuR 3DNA (GL-233) showed
extended survival of mice treated with GL-233 (49 days) compared to control animals
(31 days). GL-233 is being investigated as a complementary approach to current,
active therapies for ovarian cancer for overcoming drug resistance and
inhibiting tumor growth.
SUMMARY & FUTURE DIRECTIONS
Genisphere’s 3DNA platform
is composed entirely of noncoding DNA assembled through the sequential
hybridization of single strands of DNA into a network of double-stranded nucleic
acid having a controlled architecture, and multiple attachment sites for drug
and targeting molecules. The flexibility of the 3DNA platform enables targeted
delivery applications in gene delivery, biologics, small molecules, and RNAi
therapeutics. Through collaboration and on its own accord, Genisphere has
advanced several 3DNA-based lead compounds, and seeks additional partnerships
for development of clinically relevant programs. For example, Genisphere
recently announced a collaborative research agreement with the University of
Pennsylvania for photodynamic therapy (PDT), in which 3DNA specifically
targeted to breast cancer cells will be used to deliver photosensitizing drug. PDT
is a complementary treatment option for early stage cancer. After tumor tissue
is surgically removed, photosensitizing drugs are administered and activated by
visible light to destroy any remaining cancerous cells. The delivery of PDT to
the entire surgical field is essential, thus selective photosensitizer accumulation
in diseased cells is necessary to avoid therapy-limiting damage to normal
tissues. On the industrial side, Genisphere recently publicized a research and
option to license agreement with MedImmune, the global biologics research and
development arm of AstraZeneca. The partners will develop 3DNA nanocarriers
using up to six of MedImmune’s oncology molecules.
Genisphere continues to
explore utility of 3DNA in versatile applications. Future areas of interest
include CRISPR gene-editing strategies for oncology and neurotherapeutics based
on multi-targeted 3DNA nanostructures. With the emergence of new translational
tools in medicine and the growing need for individualized care for patients,
the 3DNA platform provides just the right amount of flexibility to rapidly
adapt to patient needs while maintaining high therapeutic efficacy and little
to no toxicity, ultimately enabling both individualized care and combination therapy.
1. Bottini M, et al.
Targeted nanodrugs for cancer therapy: prospects and challenges. J Nanosci
2. Frank D, et al.
Overview of the role of nanotechnological innovations in the detection and
treatment of solid tumors. Int J Nanomedicine. 2014;9:589-613.
3. Sanna V, et al.
Targeted therapy using nanotechnology: focus on cancer. Int J Nanomedicine.
4. Nilsen TW, et al.
Dendritic nucleic acid structures. J. Theoretical Biology. 1997;187:273-284.
5. Getts RC, Muro S.
DNA-based drug carriers: the paradox of a classical “cargo” material becoming a
versatile “carrier” to overcome barriers in drug delivery. Current
Pharmaceutical Design. 2016;22(9);1245-1258.
6. Muro S. A DNA device
that mediates selective endosomal escape and intracellular delivery of drugs
and biologicals. Advanced Functional Materials. 2014;24:2899-2906.
7. Gerhart J, et al.
Reducing posterior capsule opacification by eliminating myonog cells using 3DNA
nanocarriers and the G8 antibody. Poster presented at The Association for
Research in Vision and Ophthalmology annual meeting, May 2015. Available at: http://genisphere.com/sites/default/files/Reducing%2520Posterior%2520Capsule%252Opacification%2520by%2520Eliminating%2520MyoNog%2520Cells%2520Using%2523DNA%2520Nanocarriers%2520and%2520the%2520G8%2520Antibody.pdf.
8. Huang Yu-Hung, et al.
Delivery of therapeutics targeting the mRNA-binding protein HuR using 3DNA
nanocarriers suppresses ovarian tumor growth. Cancer Research. 2016;76(6);1-11.
C. Getts, PhD, is the Vice President of Research and Development and CSO at Genisphere.
Since joining the company 22 years ago as a Senior Research Scientist, he has actively
developed outside relationships with project collaborators and potential partners
in the biotech research and business communities for work on original and
custom products and technologies. He has led the development of Genisphere’s 3DNA
nanotechnology and IP portfolio as a signal amplification platform for
improving sensitivity in life science and diagnostic assays (for microRNA and
mRNA) and more recently as a pre-clinically validated targeted delivery
platform for cancer, cardiovascular, and central nervous system indications.
Dr. Getts has over 25 publications to his credit and continues to publish
peer-reviewed manuscripts and review articles. He has more than 15 issued patents
domestically and internationally and more than 35 submitted patent applications
internationally. In recent years, Dr. Getts has been focused on drug discovery
and the pre-clinical development of Genisphere’s 3DNA platform for both
Genisphere and partner pharmaceutical company Lead candidates, leading to the development
of an extensive dataset in support of this work.
Bowers is the Marketing Director at Genisphere. Since joining the company
in 2002, she has supported product launches, provided technical and sales
support to a global base of customers, and guided corporate communications. She
serves as project coordinator for Genisphere's collaborations with academic
institutions for targeted drug delivery.