Forty-five years ago, an article in the Journal of Molecular Biology detailed the strategy and technical know how involved in creating an antisense oligonucleotide for the first time. It took another 20 years of work until a company received clearance from the FDA to conduct the first clinical trial for an antisense product candidate. That company was ISIS Pharmaceuticals (now named Ionis Pharmaceuticals), and the product was ISIS 2105 (afovirsen).Unfortunately, ISIS-2105 did not prove successful and the program was halted in Phase 2 due to a lack of efficacy. However,while this and other early efforts to develop antisense therapeutics failed, they demonstrated the potential of the technology and effectively launched the antisense revolution.

There are several types of antisense – short interfering RNA (siRNA), single-stranded DNA (ssDNA), and anti-microRNAs – each with unique attributes that lend themselves to specific applications. At Bio-Path Holdings, we fabricate our antisense oligonucleotides with single-stranded DNA using our platform technology, DNAbilize®.

As with any antisense approach, our goal is to target and shut down proteins that are over-expressed in diseases like cancer. Our candidates are differentiated from those in development at other companies by the type of modification to the antisense molecule and the method by which it is conveyed to its target cell.


Technologies have evolved over several years that have vastly improved the stability of oligonucleotides, allowing them to escape degradation by DNA-cleaving enzymes during circulation.2Unfortunately, as often happens in the first efforts to develop novel technologies, serious limitations have been identified in these first- and second-generation technologies. Specifically, it has been shown that phosphorothioate oligonucleotides – a common first-generation modification in which an oxygen atom has been replaced with a sulfur atom – activate the complement system causing thrombocytopenia.3

The mechanism by which phosphorothioates induce this complement cascade has not been fully elucidated, but it is theorized that they bind to heparin-binding or similar proteins that interact with protective polyanions on cell surfaces.By so activating this complement cascade, phosphorothioate oligonucleotides trigger an inflammatory reaction designed to trigger the clearance of foreign bodies.

Second-generation modifications that employ 2’-O-methyl (OMe) and 2’-O-methoxy-ethyl (OMOE) additions often cause hepatotoxicity due to their propensity to accumulate in the liver, as well as vascular inflammation and some instances of idiopathic thrombocytopenic purpura, a rare clotting disorder.5


Figure 1Bio-Path Holdings has sought to overcome these problems through a proprietary platform that offers a unique solution to both DNA stabilization and delivery into the cell. With a neutral charged (or uncharged) DNA backbone using ethoxy groups in place of simple standalone oxygen in the phosphate backbone, the oligonucleotide is protected from nucleases without creating unwanted serum-protein interactions. It is also slightly hydrophobic, allowing it to incorporate readily into the lipid bilayers of a neutral uncharged lipid nanoparticle, as seen in Figure 1. Incorporation into the lipid bilayers (as opposed to the hydrophilic core or the outer surface) allows for tight association and safe delivery through the body. The lack of surface charge on the lipid nanoparticle means that cell membrane perturbations and serum protein binding that would cause steric hindrance to uptake is avoided. The nanoparticles are endocytosed into cells where the oligonucleotide is released and has therapeutic effect.

Lipid nanoparticle encapsulation of oligonucleotides has been attempted before. Initially, research into this delivery concept focused on cationic lipids because they have an overall positive charge, which would be attracted to the negative charge of oligonucleotides and to the cell membrane, enhancing cellular uptake and delivery of DNA. The first generation of cationic lipids required conjugation to a “helper” lipid, dioleoyl phosphatidylethanolamine (DOPE), which destabilizes the endosome compartment so that the nanoparticle’s nucleic acid cargo can be delivered into the target cell’s cytoplasm. Unfortunately, these cationic lipid complexes were shown to have poor stability and to absorb serum proteins in circulation, which in turn diminished the efficiency with which DNA was transferred into cells. Non-specific toxicity to cell membranes was also increased in these cationic lipid complexes.6

To our knowledge, Bio-Path is the only company to have made significant improvements in both DNA stabilization and oligonucleotide delivery. In our preclinical and clinical trials to date, we have observed no dose-limiting toxicities either from the nanoparticles or the unique ethyl modifications we employ. The DNAbilize system therefore appears to be one of the most successful ssDNA oligonucleotide therapeutics in trials today for blood cancers. We have successfully employed this DNAbilize system in the development of prexigebersen (formerly BP1001), our lead candidate for the treatment of chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML), as well as various solid tumors such as breast and ovarian cancers.

Prexigebersen is designed to inhibit-GRB2, which is highly expressed in leukemic cells and cancers with activated tyrosine kinases (eg, EGFR, BCR-ABL, FLT3, KIT). It has been shown to act as a bridge between the activated kinase and the RAS/RAF/MEK/ERK and RAS/PI3K/AKT pathways, and is critical to the survival of many cancer cells. GRB2 is a protein that does not have enzymatic activity, putting it in a class of targets historically considered to be “un-druggable.”

In preclinicalin vitrostudies, prexigebersen was effective in inhibiting the proliferation of BCR-ABL-positive leukemic cell lines as well as breast cancer cell lines that overexpress EGFR or ERBB2 leading to decreased ERK or AKT activation.7Prexigebersen was also effective in inhibiting FGF-induced motility of breast cancer cells.6

In vivopharmacology studies showed that prexigebersen was widely distributed throughout the body with a tissue half-life of 2-to-3 days. Mice and rabbits tolerated intravenous injections of prexigebersen well, with no evidence of impaired renal or hepatic functions; and blood clotting time was normal, suggesting no activation of complement cascade. Importantly, in rodents, prexigebersen was shown to distribute to many organs throughout the body, including liver, spleen, and bone marrow, where leukemia manifested; which provided us with confidence that prexigebersen would be effective in targeting this cancer.

In preclinical efficacy studies, mice with BCR-ABL-positive leukemia xenografts that were intravenously administered with prexigebersen twice a week showed a marked improvement in survival, with 80% of such mice surviving 32 to 44 days longer than control mice. Based on the positive safety and efficacy seen in preclinical models, the company entered human clinical studies.


Prexigebersen completed a Phase 1 clinical trial as a monotherapy to treat patients with AML, CML, or myelodysplastic syndromes (MDS). The primary goal of this initial study was to assess the safety and maximum tolerated dose (MTD), as well as to further define prexigebersen’s pharmacokinetics. One patient of 18 evaluable patients reported a treatment-related adverse event that was determined to be a result of treatment with a concomitant therapy. No other dose-limiting toxicities nor MTD was identified. Notably, nine of the 18 evaluable patients experienced at least a 50% reduction in peripheral or bone marrow blasts compared to baseline measures. Two patients with treatment-resistant CML had particularly impressive responses to treatment, experiencing a reduction in circulating blasts from 89% to 12% in one patient and from 24% to 7% in the other.8

Figure 2A subsequent Phase 1b trial in six elderly refractory and relapsed leukemia patients was also conducted to assess the activity of prexigebersen in combination with low-dose Ara-C (LDAC). Five of the six patients enrolled experienced a clinical response, as seen in Figure 2, with complete responses (CR) observed in three patients and partial responses (PR) in two.4Again, no MTD was identified.

The evidence that prexigebersen has been well tolerated in these studies, and that no MTD has been reached to date, leads us to hypothesize that patients may be able to remain on prexigebersen treatment longer in combination with more aggressive anti-cancer drugs that are administered at low or moderate doses to avoid increasing toxicities.

Prexigebersen’s success in targeting GRB2 stands in stark contrast to earlier industry efforts to target this gene. GRB2 is a highly sought molecular target, as it is implicated in cell cycle progression and angiogenesis, both of which are processes required for the spread of solid tumors and blood cancers. However, to date, we are not aware of any successful attempts to inhibit GRB2 either using antisense or small molecules directed to the GRB2 protein.

We also employed DNAbilize to develop a second candidate, BP1002, which targets the BCL-2 gene, which codes for a family of proteins that promote cellular survival and inhibit apoptosis. BCL-2 is highly expressed in aggressive non-Hodgkin’s lymphoma (NHL) as well as other cancers. In in vitro studies, BP1002 induced greater than 50% inhibition in 11 of the 15 aggressive NHL cell lines, including diffuse large B-cell lymphoma, mantle cell lymphoma and Burkitt’s lymphoma. In two invivo studies, 87% of mice treated with BP1002 survived until the end of the study period, compared to none of the control mice.9

BP1002 has been well tolerated in the animal studies we’ve conducted to date. Recently, for example, we completed a dose-ranging study that showed minimal impact of BP1002 on platelets, white blood cells, kidney function, or liver enzymes with any of the three doses we tested. As you can see in the bar charts in Figure 3, side effects from BP1002 were similar to untreated control mice.

Figure 3

This was an important finding, as earlier industry efforts to target BCL-2 have been hampered by lack of efficacy or significant dose-limiting toxicities, such as thrombocytopenia.10Despite these setbacks, there remains great interest in the scientific community in targeting BCL-2, as it codes for a family of proteins that can promote cancer cell survival and generate resistance to chemotherapeutics. If BP1002 succeeds in blocking the production of these proteins while maintaining a strong safety profile, it could be an important therapeutic option for oncologists. Not only might it have utility as a monotherapy, but the potential exists that it could restore sensitivity to cancers that had previously been resistant to treatment, opening up an array of treatment combinations. We are planning to initiate a Phase 1 trial to test BP1002 in patients with relapsed/refractory lymphoma.


Beyond the treatment of cancer, we believe DNAbilize has the potential to generate antisense therapies against other non-enzyme targets currently considered to be un-druggable. We believe this to be a significant business development opportunity for Bio-Path and a significant R&D opportunity for the biotech industry, as non-enzymes make up a majority of the proteome. Such protein targets include those involved in structural integrity of cells, signaling pathways, subcellular transport, transcription, translation, and other critical functions. Indications associated with errant non-enzyme proteins include autoimmune and inflammatory disorders, such as inflammatory bowel disease, asthma, and atherosclerosis; neurological and developmental disorders, such as Coffin-Siris syndrome, focal segmental glomerulosclerosis (FSGS), Rett Syndrome, Cornelia de Lange Syndrome, and Roberts Syndrome; cardiovascular diseases, including arrhythmia, fibrosis, and hypertrophy.

As is the case with cancer patients, individuals with autoimmune, neurological, and cardiovascular diseases and conditions often have additional co-morbidities and are taking several medications to control symptoms. We believe that the low level of off-target toxicities seen with our two candidates to date suggest that DNAbilize products should not elicit any drug-drug interactions with patient’s ongoing treatment regimens. Moreover, it should be able to be combined with broad spectrum pharmaceuticals to increase their therapeutic effect without increasing side effects.

We are engaged in discussions with corporate and academic research organizations for the out-license of rights to the many potential development opportunities in the DNAbilize platform in specific non-cancer indications, and will continue to explore these and other business development opportunities. Companies that are interested in exploring collaborations or licensing agreements for DNAbilize should contact our business development office


  1. Cowsert LM, Fox MC, Zon G, Mirabelli CK. In vitro evaluation of phosphorothioate oligonucleotides targeted to the E2 mRNA of papillomavirus: potential treatment for genital warts. Antimicrob Agents Chemother. 1993;37(2):171-177.
  2. Mansoor M, Melendez AJ. Advances in antisense oligonucleotide development for target identification, validation, and as novel therapeutics. Gene Regul Syst Bio. 2008;2:275-295.
  3. Wang G. Deep dive on chemistry and safety profiles of therapeutic oligonucleotides.Jefferies Analyst Report. December 7, 2016.
  4. Brown DA, et al. Effect of phosphorothioate modification of oligodeoxynucleotides on specific protein binding. J Biol Chem. 1994;28;269(43):26801-26805.
  5. Frazier KS. Antisense oligonucleotide therapies: the promise and the challenges from a toxicologic pathologist’s perspective. Toxicologic Pathology. 2015;43:78-89.
  6. Ozpolat B, Sood AK, Lopez-Berestein G. Liposomal siRNA nanocarriers for cancer therapy. Adv Drug Deliv Rev. 2014;66:110-116.
  7. Ashizawa AT, Cortes C. Liposomal delivery of nucleic acid-based anticancer therapeutics: BP-100-1.01. Expert Opin Drug Deliv. 2015;12(7):1107-1120.
  8. Ohanian M, et al. Phase I study of BP1001 (Liposomal Grb2 Antisense) in patients with hematologic malignancies. Poster presented at 2016 ASCO.
  9. Ashizawa A, et al. Activity of Bcl-2 antisense therapeutic in aggressive non-Hodgkin’s lymphoma,. Poster presented at 2017 AACR.
  10. Lindsey ML, et al. Killing two cells with one stone: pharmacologic BCL-2 family targeting for cancer cell death and immune modulation. Front Pediatr. 2016;4:135.

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NeilsenPeter Nielsen co-founded Bio-Path Holdings, a public biotechnology company developing targeted oncology therapies,and currently serves as Bio-Path’s President, Chief Executive Officer, Chief Financial Officer, and Chairman of the Board of Directors. At the time of Bio-Path’s establishment in 2007, Mr. Nielsen licensed technology and targets from the University of Texas MD Anderson Cancer Center and coordinated preclinical development, optimization, and manufacturing of Bio-Path’s lead product, prexigebersen. Over the next 10 years, Mr. Nielsen led the clinical advancement of prexigebersen into Phase 2 studies, the introduction of additional pipeline candidates, and the company’s public market debut. Prior to co-founding Bio-Path,Mr. Nielsen has worked with several other companies, leading turnarounds and developing and executing on strategies for growth. He earned degrees in Engineering, Mathematics, and an MBA in Finance from the University of California at Berkeley.

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