This review represents a series of medical hematology-oncology papers by Gordon, Chawla and Hall, composed with the following intents and purposes: (i) documenting significant milestones in Tumor Targeting and Cancer Gene Therapy for the postmodern biomedical and veterinary research communities, (ii) honoring forthright principles of scientific responsibility, regulatory oversight, and informed consent, which serve to protect both human and companion animal health, and (iii) translating FDA-qualified, updated methods & compositions (CMC and cGMP insights) to veterinary medicine, in light of recent critical analyses, hence gains, in long-term cancer-free survival data achieved in humans. The focus is on tumor-targeted, injectable gene therapy vectors and molecular approaches developed by Gordon and Hall at USC / Children’s Hospital Los Angeles-validating “Pathotropic” (disease-seeking) tumor targeting Avant la Lettre. The discussion provides clinical insights into the cellular and molecular mechanisms of both tumor-targeting and tumor-eradication, which led to the formal return of Rexin-G as DeltaRex-G from industrial desuetude to cGMP bioproduction in 2018-2019, with FDA Expanded Access, under a decidedly academic non-profit banner, in accord with historic U.S. “Right-to-Try” legislation. In the spirit of academic fellowship and the interests of a rightfully concerned society, this article provides a molecular (cellular and genetic) “Rosetta Stone” for the responsible translation and biotechnology transfer of these clinically validated U.S. FDA-evolved Pathotropic (i.e., disease-seeking) tumor targeting Methods and Compositions safely to Veterinary Medicine as preclinical safety and efficacy studies in mice, rats, rabbits, minipigs and monkeys were required by the USFDA prior to authorization to use DeltaRex-G in first-in-human clinical trials.
Abbreviations: Targeted Gene Delivery; Cell Cycle Control; CCNG1; DeltaRex-G; DeltaVax
“For love of companion animals” and a deep respect for the distinguished professors and colleagues, who guided a veterinary school graduate student, now a Ph.D. in Physiology and Biophysics, and a physician scientist through the primordial muck of the evolutionary tidepool; through the shapeshifting RNA Worlds of biochemical cause and effect ; through the illustrious Kingdoms of the Virus (Virae), the Amoeba (Protozoa), and the lowly Planarian* [2-5], to this prescient pinnacle of mammalian cell biology, cancer biology, regulatory biology, and stem cell epigenetics: a pinnacle upon which the hard-won, hard-core digital logic (hence molecular cybernetics) of site-specific, “Proline-directed Protein Phosphorylation” [6-8] accompanied by phosphorylation-specific-proyl (cis-trans) isomerization [9,10] can be viewed as fundamental to understanding medical disorders ranging from dementias of the aging human brain to cell cycle regulation, including the clinical management of metastatic cancers.
In this invited translational research paper, we highlight biomedical, bioethical, and RAC/FDA established principles which served to guide our research into orthopedic limb-salvage, tissue regeneration, wound healing, stem-cell-capture, and metastatic cancer control—from the prestigious think-tanks, operating theaters, ballrooms, courtrooms, and conference tables of modernday princes, royals, and occasional knights**; through the meanstreets of contrarian academics; with it, departmental tycoons and manifold influences of a global pharmaceutical industry; through seasons of testing, trials, and travels in diverse places to this rarefied Vista of Medical History wherein (i) the primal biochemistries, (ii) the molecular genetics, and (iii) the physiological targeting technologies of our shared medical mission have been scientifically validated, affirmed, and restored to rightful intellectual sponsors upon critical analysis of long-term cancer-free survival outcomes. In the spirit of academic fellowship and the interests of a concerned society, this paper aims to provide a molecular / cellular / genetic “Rosetta Stone” for the responsible translation and bio-technology transfer of a broad-spectrum Anti-Cancer Gene Therapy platform with Pathotropic (Tumor-Targeted) Gene Delivery methods and compositions safely to Veterinary Medicine.
*Malignant Reticuloma (a primordial “leukemia”) of the Planarian (archived at the U.S. Smithsonian Institute) is considered among the simplest animal models demonstrating chemical cocarcinogenesis [2-5]: that is, initiation and promotion of cancer.
**Prof. Philip Cohen (now Sir Philip Cohen), principle investigator of the renowned MRC Protein Phosphorylation Unit, University of Dundee, Scotland, graciously invited veterinarianscientist Dr. Philip Vulliet and postdoc Frederick Hall, Ph.D., through a NATO-sponsored collaboration, to test / substantiate Dr. Hall’s original deduction / hypothesis (of site-specific “proline-directed” protein phosphorylation) with hard-core radio-sequencing of the NGF(nerve growth factor)-sensitive Synapsin I site (a target substrate) by sequential Edman degradation .
Proline-directed protein phosphorylation would ultimately expand in scope (and prominence of proline-directed protein kinase regulatory partners) to delineate the biological transduction of vital extracellular signals through receptor-mediated relay mechanisms, identifying linear cascades of digital (reversible, post-translational) phosphotransferase activities (on/off cybernetics) governing the activation and expression of “Executive proto-Oncogenes” in animal stem cells, leading to discovery / characterization of the commanding CCNG1 (Cyclin G1) proto-oncogene and the clinical development of DeltaRex-G (Formerly Mx-dnG1, dnG1, Rexin-G; Figure 1). Table 1 [6-51] highlights epochs of discovery in biomedical research, which flourished at USC / Children’s Hospital, Los Angeles, California and further developed through preclinical research (in mice, rats, rabbits, pigs, and monkeys) into far-reaching clinical applications.
Targeting the Executive Proto-Oncogenes of Animal Stem Cell Competence
Mechanistic Focus on the Pivotal, Commanding Cyclin G1 / Cdk / c-Myc / Mdm2 / p53 Axis
The mechanisms of proline-directed protein phosphorylation governing cell cycle activation, entry, metabolic growth, progression, DNA replication, and cell division are highly conserved. Indeed, the identification of core checkpoint control mechanisms dependent on coordinated cyclin-dependent kinase (Cdk) activities (i.e., Cyclins) and phosphatase activities (i.e., PP2A-regulators) has been recapitulated in what seems like “a zillion” moleculargenetic screens . Enter the human CCNG1 (Cyclin G1) proto- Oncogene which was identified by molecular genetics [21,53], and characterized functionally in terms of gene expression / knockout in human osteosarcoma cells , which often lack TP53 (p53) tumor suppressor function entirely.
What was once considered controversial, in terms of Cyclin G1 function in neoplastic transformation and oncogenesis vis-àvis p53-mediated tumor suppression [54-57], has been clarified by direct experimentation, informative preclinical studies, and pioneering “tumor-targeted” gene therapy applications complete with clinical validation of (i) histological, (ii) cellular, and (iii) molecular mechanisms-of-action [36-40], providing (iv) long-term, cancer-free survival outcomes [46-51]. Figure 2A reveals with clarity the potential diagnostic and therapeutic insight the hardcore structure/function relationships described operationally as the Cyclin G1-Cdk / cMyc / Mdm2 / p53 Axis the axis mundi of Stem Cell Competence-which links CCNG1 expression and the resulting Cyclin G1-dependent phosphotransferase activities to stem cell activation and oncogenic transformation (see Figure 2B), revealing a potential for broad-spectrum clinical utility in the management of advanced metastatic cancers, based on bioactive tumor targeting, and hardwon molecular-genetic control of this commanding oncogenic driver.
Stem Cell Activation, leading to stem cell growth, DNA synthesis, and mitotic cell division, is orchestrated by the expression and biochemical functions of “Cyclins,” a conserved family of executive regulatory proteins that function as intracellular targeting-subunits for their cognate cyclin-dependent, proline-directed protein kinase (CDK) partners. The Cyclin Proteins, named alphabetically in order of their discovery and molecular cloning in animal cells, are defined structurally by a characteristic Cdk-interacting domain, the so-called “cyclin-box.” Cyclins are divided operationally into subfamilies as either (i) “Canonical Cyclins” (Cyclins A1 to E2), which bind/ target/activate Cdks of the cell division cycle; (ii) “Transcriptional Cyclins” (e.g. Cyclin H) which are involved in the regulation of RNA polymerase; and (iii) “Atypical or Orphan Cyclins,” including CCNG1 (Cyclin G1), which is considered both canonical: Cyclin G1/Cdk heterodimers target/phosphorylate/stabilize the cMyc competence factor, and transcriptional: the p53 tumor suppressor and the Cyclin G1 Survival factor normally cooperate in mediating genomic stability and DNA damage repair in somatic cells, integrating cell survival and cell death decisions during the process of cell differentiation [57-60]. Indeed, the Cyclin G1-associated p18 FX3 zinc-finger (DNA-binding) protein first identified at USC / CHLA in a molecular-genetic screen for human Cyclin G1-interacting proteins -was re-named “Hamlet” in Britannia and Spain  upon demonstrations that this transcriptional coactivator provides an important link between extracellular (MAPK) signaling pathways , CCNG1 gene expression, and p53-dependent life-ordeath cell-fate decisions: Transcriptionally p18 Hamlet regulates (i) p53-dependent pro-apoptotic responses to DNA damage-inducing agents , as well as natural (ii) epigenetic mechanisms regulating gene activation and muscle stem cell functions  located at the headwaters of tissue repair, myogenesis, and muscle stem cell differentiation  hence, potential for limb regeneration.
The Cyclin G1-Cdk / cMyc/ Mdm2 / p53 Axis is illustrated in Figure 2A: expression of CCNG1 (Cyclin G1) is controlled by a number of suppressive RNA species, including microRNAs miR- 122, miR-23b, miR-27b, and long non-coding RNA LINC01494 [65- 70], the loss of which results in the upregulation of CCNG1 (Cyclin G1) and the Cyclin G1/PP2A-dependent activation of the Mdm2 oncoprotein, a negative regulator of p53 . The expression of the Cyclin G1 onco-protein physically binds and targets a cellular cyclin-dependent kinase partner (Cdk1, Cdk2, or Cdk5) to the cMyc Competence Factor [71,72], whereby the phosphorylation of cMyc at Ser-62 [73-75] “stabilizes” this key oncogenic transcription factor, thereby activating gene expression through “E-box” promoter elements of particular genes associated with stem cell proliferation, maintenance of pluripotency, progression to cancer, and metastasis.
Mutational loss of the tumor suppressors BRD7 and/or TP53 [76,77] upregulate CCNG1 oncogene (Cyclin G1) expression, amid increasing genetic instability, as does the molecular suppression of miR-122 by the long non-coding RNA named HOTAIR, a novel type of “RNA-oncogene,” whose expression is linked to cancer metastasis [78,79]; as does viral subversion of the CCNG1 gene locus by the hepatitis B virus (HBV) X-protein (HBx), whereby direct suppression of miR-122 expression “unleashes” CCNG1 (Cyclin G1) expression, which drives hepatoblastoma cell proliferation .
Molecular Engineering and Precision Delivery of the DeltaRex-G “Silver Bullet:”
A Dominant-Negative Cyclin G1 (dnG1) Inhibitor for Broad Spectrum Anti-Cancer Gene Therapy
The executive role of CCNG1 expression in governing the activation and stability of cMyc and Mdm2 core onco-proteins governing Stem Cell Competence is revealed in the bifurcation of structure-function relations attributable to the Cyclin G1 protein, as illustrated in Figure 3A. The corresponding structure of the dominant-negative (dnG1) killer gene, a Cyclin G1 Inhibitor, (aka dnG1 Silver Bullet) developed for advanced metastatic cancers is shown in Figure 3B. In designing the cytotoxic “dnG1” construct, Hall and Gordon purposefully eschewed molecular antisense constructs and point mutations in the “cyclin box” domain, which were indeed insightful in terms of elucidating molecular mechanisms and essential functions; however, such constructs are considered nonoptimal in terms of blocking robust signal transduction pathways wherein the catalytic turnover rates (kcat) of protein kinase and phosphatase reactions are measured in seconds, and resulting phosphorylation-dependent stabilizations, assemblies, and enzyme activities of key regulatory proteins have wide ranging cellular implications involving cell life-or-death decisions.
The dominant-negative dnG1 expression construct embodied in DeltaRex-G, is delivered intravenously yet selectively to tumors via a physiologically-active Pathotropic (disease-seeking), tumorsurveilling gene expression vector [30-40], which performs clinically as a broad-spectrum anti-cancer agent: blocking Cyclin G1-dependent survival pathways in chemo-resistant cancer cells derived histologically from differing germ layers (e.g. pancreatic cancer, soft tissue sarcomas, osteosarcoma, and breast cancer): enforcing apoptotic death in cancer cells and cancer stem-like cells, blocking tumor-associated neo-angiogenesis, resolving lymphatic metastasis, and restoring and/or enhancing anergic tumor immune responses.
The Survival Value of Bio-Active Tumor Surveillance
The Clinical Performance of DeltaRex-G a retroviral expression vector bearing an inhibitory construct of Cyclin G1 represents a milestone in Medical Nanotechnology , as well as Cancer Gene Therapy and Immunotherapy. Documentation of physiological tumor targeting, apoptotic mechanisms-of-action, dose-dependent bio-pharmacology, primary tumor eradications, metastatic cancer eradications, and dose-dependent survival benefits have all been demonstrated formally, within the guidance and confines of regulatory oversight, leading to USFDA orphan drug and fast track designations for pancreatic cancer, soft tissue sarcoma and osteosarcoma, and PhFDA accelerated approval for all solid malignancies deemed refractory to standard chemotherapies in 2007 [36-40,82,83], and Expanded Access for DeltaRex-G for solid malignancies such as pancreatic cancer, sarcoma, breast and prostate cancer, cholangiocarcinoma, NSCLC and Emergency Use Authorization for Severe COVID-19 [46-51] in 2020.
As defined by the NCI, Retrovector Encoding Mutant Anti- Cyclin G1, DeltaRex-G, is a replication-incompetent, pathotropic, tumor / matrix (collagenous SIG)-targeted, retroviral vector encoding an N-terminal deletion mutant form of the Cyclin G1 gene with potential antineoplastic activity. Under the control of a hybrid long-terminal repeat/cytomegalovirus (CMV) promoter, retrovector encoding mutant anti-cyclin G expresses the mutant cyclin G1 construct, resulting in disruption of tumor cell cyclin G1 activity and decreased cellular proliferation and angiogenesis. This agent preferentially targets collagen (SIGs) of the tumor matrix because of the incorporation of the collagen-binding domain of von Willebrand factor (vWF) on the retrovector surface. Exploiting the collagen-targeting mechanism of vWF permits delivery of the retrovector to tumor sites where angiogenesis and collagenous matrix exposure occur (NCI Thesaurus).
DeltaRex-G represents a potential cross-species “dnG1 Silver Bullet” for advanced metastatic cancers: Enforced expression of dnG1, a Cyclin G1 Inhibitor, blocks hyper-competent survival pathways, restores lost tumor suppression (via enforced apoptosis), and disrupts core oncogene addictions characteristic of cancer stem cells (Figure 3A). Designed to function physiologically (indeed, pro-actively) amid the turbulence, flow, shear forces, filtration, immune inactivation, and manifold dilution factors encountered within the systemic circulation, the (anaplasia, pan-Col) SIGtargeted DeltaRex-G expression vector seeks-out and accumulates selectively in cancerous lesions—including inoperable primary tumors, remote metastatic lesions, and even lymphatic drainage— demonstrating objective, quantitative, dose-dependent anti-cancer activities, including tumor necrosis, overt anti-angiogenesis, tumor eradications, and lymphatic clearance; leading to clinical remissions with survival benefits, as summarized in the following “Bullet Points” describing the targeted anti-cancer bioactivity of DeltaRex-G with histopathological precision:
1. Delta-Rex-G targets proliferative tumor cells, cancer stem cells, and tumor-associated fibroblasts;
2. Delta-Rex-G targets angiogenesis: tumor-associated stroma, endothelial cells, and immune cells;
3. DeltaRex-g exhibits excellent clinical safety, dose-dependent responses, single-agent-efficacy (monotherapy);
4. DeltaRex-G exhibits broad-spectrum bioactivity in advanced, chemo-resistant metastatic cancers;
5. Notably, DeltaRex-G penetrates and ‘clears’ the lymphatic system of metastasis, over time; and
6. Delta-Rex-G restores immunity while eliminating chemotherapy-resistant cancer stem-like cells.
Piloted by a unique (tumor-seeking) navigational guidance system, the DeltaRex-G expression vector physically delivers: it enters, downloads itself (reverse transcribes its RNA-to-DNA) and enforces the expression of the cytocidal “dnG1 Silver Bullet” in the proliferative tumor cells of primary and metastatic lesions: in transformed cancer cells, proliferative tumor neo-vasculature, myofibroblast-like stromal cells, and tumor immune cells arresting tumor growth (in size and metabolic activity), arresting tumor neoangiogenesis, and restoring / enhancing both innate and adaptive immune responses [36-40], including the phagocytic clearance of tumor debris [37,84], as documented by a detailed histological and cytological analysis of immune cell trafficking in the tumor microenvironment of DeltaRex-G (Cyclin G1 inhibitor)-treated tumors [48,85-88].
“The realization that the gene delivery function (i.e., cytocidal activity) of pathotropic nanoparticles remains active as it penetrates metastatic disease within sentinel lymph nodes and does not disrupt but appears to work in concert with the immune system, reaffirms the potentiality of future cancer vaccinations in situ, using this targeted gene delivery system bearing a cytokine gene.” .
The ability of DeltaRex-G to work in concert with the cancer patient’s own immune system in managing tumor eradication encouraged the development of a New Generation of Gene Delivery Vectors, which “target metastatic cancers from the inside” , enabling the conception and clinical development of The Genevieve Protocol: an innovative Dual Targeted Approach to Cancer Gene Therapy / Immunotherapy. The protocol introduced a second treatment (post-dnG1) as adjuvant immunotherapy, deploying DeltaVax (formerly Reximmune-C), a pathotropic tumortargeted cytokine vector, which pro-actively delivers the immunostimulatory GM-CSF gene (expressed, pulsed) to ‘residual tumor cells’ to enhance localized tumor immune responses and improve treatment outcomes thereby demonstrating prolonged survival times and advancing the medical concept of Personalized Cancer Vaccinations (in situ auto-vaccinations) as a realistic and nowachievable clinical goal [41-43].
Moreover, Hall and Gordon subsequently adapted pathotropic (disease-seeking) targeting technologies to therapeutic proteins to enable the targeted delivery of monoclonal antibodies (mAbs) with a series of synthetic targeting peptides (named Onco-aptamers) [44,45], designed to bind physically (upon mixing) and pro-actively deliver existing off-the-shelf FDA-approved humanized monoclonal IgGs to tumors: including anti-tumor cell mAbs, anti-angiogenesisacting mAbs, and / or the popularized immune checkpoint inhibitors, which continue to elicit serious immune-related adverse events , when left un-tethered. Pathtropic Targeting was developed by Hall and Gordon et al. at the turn of the century, a time in the history of molecular-genetic medicine when the collective opinion of the pharmaceutical industry was decidedly negative, having harvested a clutch of U.S. taxpayer-funded research programs, intellectual properties, and fledgling biotechnology companies, with nary a clinical gene therapy treatment nor product in sight. According to an astute industry analysis at the time:
Researchers Get a Dose of Reality as Logistics Stymie Gene Therapy
by Robert Langreth and Stephen Moore, The Wall Street Journal, 1999
“From the Beginning, the therapy’s main difficulty has been a logistical one: how to deliver enough healthy genes to the appropriate site and get them to stay there long enough to cure or alleviate a disease” .
It is in this context of strained academic and scientific responsibilities that the inspired research collaboration of Hall and Gordon advanced as a concerted and enduring medical mission: research in biological signal transduction, stem cell biology, hematology, oncology, molecular genetics, and translational research (Table 1)—working together (i) to capture primitive P4 mesenchymal stem cells for wound healing and tissue regeneration, (ii) to target growth factors and other therapeutic agents in an effort to treat / vascularize / heal injured and diseased organs, (iii) to identify, characterize, and harness rate-limiting oncogenes governing stem cell competence and neoplastic transformation, and finally (iv) to conceive, reduce-to-practice, optimize, and manufacture biologically safe, pathotropic, replication-incompetent gene delivery/therapy vectors for clinical applications in oncology in accordance with the evolving expectations, scientific understandings, FDA-guidance, and “informed consent” befitting a rightfully concerned society.
Fortunately, the passage of recorded time in relation to clinical outcome has an impeccable form of sanctity. It is the clinical safety, anticancer efficacy, and survival value of repeated DeltaRex-G infusions demonstrated in the crucible of clinical oncology (in chemotherapy-resistant metastatic cancers), where investigational new drugs (INDs) and clinical protocols are contested, adverse events and cancer-free remissions are dutifully recorded [47- 51], and long-term patient follow-up is formally mandated by the U.S. FDA. In view of actual long-term cancer-free survivors now exceeding 10-12 years of cancer-free survival, in certain hard-totreat chemotherapy-resistant metastatic cancers [see 85-88] it is clear that overall survival (OS) represents a New Gold Standard for evaluating treatment outcomes of metastatic cancer therapies, which amounts to somewhat more than an academic milestone .
Critical analysis in aggregate and individual case studies over the years [92,93] has revealed molecular mechanisms of action and resulting tumor cell responses in graphic detail, as the histological processes of tumor necrosis and tumor eradication emerged: eliminating troublesome chemotherapy-resistant cancer cells and their stromal accomplices along with troublesome cancer stem cells, which cannot escape the physiological tumor surveilling function of the pathotropic DeltaRex-G nanoparticles. A recent discovery in myeloid neoplasms focused attention on CCNG1 as a protooncogene: Geneticists at Harvard University and the Mayo Clinic recently reported on a novel oncogenic gene mutation—specifically, an IGH-CCNG1 Gene Rearrangement found in a patient with chemotherapy-induced acute myeloid leukemia (AML), thus sounding alarm bells for medical oncology; and yet identifying DeltaRex-G (Cyclin G1 inhibitor) astutely as a potential new treatment for such deadly AML. According to Dr. Sheng Xiao et al. of Harvard University:
“Clinical trials with a dominant-negative CCNG1 retroviral expression vector (DeltaRex-G) showed impressive results, including several metastatic tumors being cancer-free ten years after DeltaRex-G monotherapy.... It will be interesting to evaluate how frequent the CCNG1 is overexpressed in myeloid tumors, and whether an anti-CCNG1 strategy such as DeltaRex-G is effective in treating myeloid tumors, a group of diseases typically with very poor prognosis” .
Consistent with these findings, Gordon et al. have confirmed that the CCNG1 (Cyclin G1) gene is highly expressed in numerous advanced cancers, independent of TP53 (p53), and are currently developing companion diagnostic assays to identify patients who are likely to benefit from DeltaRex-G . This molecular genetic discovery of an oncogenic breakpoint translocation within the CCNG1 gene promoter region is both clinically and mechanistically revealing: (i) it introduces and exhibits real-world molecular oncology of gene expression enhancers and (ii) it confirms that the overexpression of the Cyclin G1 is sufficient, by itself (via the Cyclin G1 Axis; see Figure 2A), to actually cause cancer: in this case, an acute and rapidly-fatal myeloid leukemia.
The complex circuitry regulating CCNG1 (Cyclin G1) expression in the presence and absence of inactivating TP53 (p53) mutations can now be examined at the level of the gene promotor: Gene promoters contain several classes of functional sequence elements (cis elements) recognized by regulatory proteins, various transcription factors, and essential components of the transcriptional machinery. Mechanistically, a particular cis-element (TCTCGCGATA motif) is found in the promoter regions of both the cMyc and CCNG1 protooncogenes : the motif appears to be critical for the expression of genes involved in enhancement of protein synthesis during the early phases of the cell division cycle. This cis-element is duplicated in CCNG1, as it is duplicated in a subset of transcription factors, translation initiating factors, and ribosome proteins involved in cell cycle activation and entry .
Another key to understanding the coordinated activation and cell-cycle regulation of cMyc, Cyclin G1, and Mdm2 proto-oncogenes [50,51] is the so-called Specificity protein 1 (Sp1), a transcription factor (family) that exerts its effects on genes containing GC-rich Sp1- binding sites within their promoters—the transcription of which mediates processes of cell growth, differentiation, angiogenesis, stem cell programming and tumorigenesis . Of clinical interest, as an adjuvant treatment, are reports that anti-cancer activity induced by oleanolic acid, a natural pentacyclic triterpene [98,99], is manifested at least in part at the level of CCNG1 (Cyclin G1) gene expression: suppressed in lung carcinoma cells by an increase in abundance of miR-122 , a natural CCNG1 (Cyclin G1) gene suppressor that is commonly lost with the progression of cancers. See Table 2 [101-108] for additional newsworthy research reports supporting the unifying concepts, themes, and biochemistries presented herein.
In Retrospect, the Year 2019 represents a modern-day annus mirabilis (a year of wonders): a year in which the U.S. Cancer Patient’s Right-To-Try legislation (now U.S. Law) empowered the restoration and return of clinical DeltaRex-G (an FDA-qualified IND) the first time in a decade; a year in which Gordon and Hall strived together to shine a collaborative light fantastic one more time upon a concerted, astonishingly-assembled, grass-roots (charitably-funded), biotechnology-enabled, University-supported, FDA-granted, Right-to-Try empowered Rescue Mission of 2019 a medical mission to resurrect the FDA-approved chemistries and manufacturing controls (CMC) needed to rescue the life-saving biotechnologies from desuetude. The genetic reconstruction, bioproduction, and recertification of the requisite plasmid DNAs, with updated CMC and approved vector cGMP  returned DeltaRex-G to the cancer clinic with FDA approval of the “Blessed Protocol” in 2019 (NCT04091295).
The BLESSED Protocol
Expanded Access for DeltaRex-G for Advanced Pancreatic Cancer and Sarcoma(s)
… ClinicalTrials.gov Identifier: NCT04091295 
It bears mentioning, that the demands for scientific integrity, like the demands for empirical truth, are severe; neither have any sympathy for unearned laurels, fame, nor bio-pharmaceutical fortunes which become like chaff the wind drives away. Therefore, due caution, scientific integrity, a protective (do no harm) posture, and a covenant of compliance is well-advised, as we hand-off this Seaworthy Primer on Pathotropic Medicine, from the FDA Centennial Celebrations of 2007  to Veterinary Medicine, with the same idealistic “clinical principles, adages, and aphorisms” that guided research progress, in our time.
Caution! A modern medicine should not be a replicating species! . At this point, a categorical distinction indeed, a moral distinction must be made between the “replication incompetent” retroviral vectors presented herein, which are constructed from inert components with no replication competent characteristics, and the infectious species of viruses that has been advocated in recent years with a disturbing epidemiological naiveté and a less than convincing scientific rationale… Hence, it bears reiterating that DeltaRex-G is the first and only systemically injectable, immunologically stealth, mechanistically defined, genetically stable, certifiably replication-incompetent gene delivery vehicle that is designed and proven to be safe and effective in the clinic .
Remembering “Alisomar-1975” International Congress on Recombinant DNA Molecules. Then and there, yonder upon the far-reaching Alisomar State Beaches of the Monterey Peninsula, 140 top bio/life-scientists and physicians, accompanied by lawyers, journalists, and government officials, gathered at the Alisomar Conference Center in Pacific Grove to voice their concerns and to recommend a voluntary moratorium on certain recombinant DNA experiments  specifically, experiments that introduced genes which could potentially transform normally innocuous microbes into cancer-causing agents or human pathogens resistant to antibiotics  more specifically, the biosafety concerns focused intently on the Simian Virus (SV40), which turns out to be “unexpectedly” dangerous in terms of human health ; yet SV40 virology was immensely useful in terms of understanding and eventually utilizing viral gene-expression mechanisms and elements safely for therapeutic gene delivery.
Ultimately, it was agreed that the research should continue but under stringent guidelines, thus marking a historic milestone for science and for public discussion of science policy, leading directly to the formation of the U.S. FDA RAC. Looking back, meeting organizer Paul Berg reflects: “There is a lesson in Asilomar for all of science: the best way to respond to concerns created by emerging knowledge or early-stage technologies is for scientists from publicly-funded institutions to find common cause with the wider public about the best way to regulate as early as possible. Once scientists from corporations begin to dominate the research enterprise, it will simply be too late” .
Pathotropic Vector Production: “Points to Consider” for a Comparative Oncology
DeltaRex-G is a tumor-targeted MLV-based retroviral expression vector (~100 nm) displaying an amphotropic (panCol) SIG-targeted envelope on its surface to seek-out anaplastic proteins exposed pathologically within the tumor microenvironment ; the DeltaRex-G retrovector encodes a cytocidal Cyclin G1 inhibitor, dnG1 (Figure 3A), under the control of a hybrid CMV/LTR promoter . The vector contains the neomycin resistance gene (neo) driven by the SV40 early promoter; neo expression is useful for certifying functional vector titers (gene transfer units/ml) and also for tracking / marking of transgene expression in vivo. However, in the case of a tumor-targeted vaccine (DeltaVax), the neo marker gene was replaced with HSV-tk thymidine kinase gene, a so-called suicide transgene for additional safety: (i) to avoid potential engraftment, (ii) to enable pharmacological control of gene expression with herpetic prodrugs, and (iii) to restrict GM-CSF expression locally within tumors .
The targeted DeltaRex-G vector specifically designed for use in humans is produced by transient co-transfection of 3 separate plasmids in HEK 293T (human 293 kidney cells transformed with SV40 large T antigen) producer cells obtained from a fully validated master cell bank [46,86-88]. The restriction of transgene expression to proliferative cells is a safety feature of the MLVbased vector, while transient transfection versus “stable” producer cells provides for greater genetic fidelity of the IND [116-119]. Moreover, the Delta-Rex (dnG1) and Delta-Vax (GM-CSF) vectors were optimized to eliminate overlapping DNA sequences within the three deconstructed plasmids  that might inadvertently recombine to generate unwanted replication-competent retrovirus (RCR): thereby providing for safer (certifiably RCR-free), sterile, clinical- grade expression vectors with consistently high titers (Figure 3B). These efforts of refinement were authorized by the U.S.FDA, which subsequently granted DeltaRex-G “Orphan Drug” and “Fast Track” status.
Biologically, the dnG1 Silver-bullet (packaged in a plasmid) literally “shoots-the- heart-out” of Animal Cell Competence that is, the Cyclin-G1/Cdk/cMyc/Mdm2/ (+/-) p53 Axis, which is conserved at the protein level from the eggs and embryology of sea urchins, frogs, fruit flies, and salamanders, to the regenerative stem cells residing within the fragile corpus of mankind. Likewise, the Molecular Biology of enforced Gene Expression, well-known to the kingdom / family vividae, has broad spectrum applications as: the same dnG1 for cats (Figure 4), and dogs, and ponies. Immunologically, however, the Veterinary Producer Cells must naturally be species-specific to exhibit circulatory and surveilling stealth, enabling repeated infusions without untoward immune adverse events. Among the various methods for delivering the three structural “blueprints”  of this “Three-Plasmid MLV-based retrovector System” Three plasmid DNAs, in precisely defined ratios to a highly transfectable “Certified producer cell line” which is rationally engineered with powerful synthetic viral elements, and yet is “certifiably-virus free” and demonstrably capable of generating “replication-incompetent” gene expression vectors for cancer gene therapy [116-119].
The final product exhibits a vector titer of 2-5 x 109 colony forming units (cfu) per milliliter, a biologic potency of 50-70% growth inhibitory activity in target cancer cells, less than 550 bp residual DNA, no detectable E1A or SV40 large T antigen, and no detectable replication competent retrovirus (RCR), in compliance with FDA recommendations for retroviral vector-based gene therapy products. The vector formulation is stored in aliquots of 60 ml in a 250 ml cryobag and kept frozen at -70° to -90°C until used. Preparation of the DeltaRex-G vector for patient administration consisted of rapid thawing of the vector in the cryobag in a 37°C water bath. The vector is thawed 15 to 30 minutes prior to infusion into the patient and given intravenously at 4 ml/per minute. All personnel who handled and disposed of the vector observed Universal Precautions compliance in accordance with the National Institutes of Health Guidelines for Research Involving Recombinant DNA molecules.
In closing, we concur with ethical and scientific appeals for a more effective, combinatorial, and comparative oncology, as championed by Thomas Efferth, whose scholarship has done much to reveal the efficacy of Artemisinin’s (selective ferroptosis) in combination with chemotherapies for both medical and veterinary oncology [120-122], presenting a positive challenge to consider the spontaneous cancers in companion animals as both vital and opportune . It is with confidence in the unity of nature and homeostasis—as reflected in these hard-won molecular, cellular, and genetic understandings that we can expect to learn more about managing cancer metastasis within ourselves, by targeting tumors and managing extended remissions in veterinary medicine.
Acknowledgments, Appreciations, and a Memoriam
The authors are grateful to the Aveni Foundation A-team Rescue Mission Members: John P Levy (deceased), Rebecca Reed, Heather Gordon, Alison Muldoon, Nicholas J. Hall, Dexter W. Gaston, Jesus Hernández and Brody’s Compassionate Veterinary Care in Monterey, California: Carmel Valley Veterinary Hospital, Dr. Aysha Taff and Dr. James Holt. Ophthalmology for Animals, Dr. Elizabeth Curto; Pet Specialists of Monterey (emergency surgery), Dr. Greg Marsolas, Dr. Samantha Frankovich, and Dr. Bill Sullenberger; Animal Cancer Center of Monterey, Dr. Teresa Artega, and to Lance Ostendorf for his generous donation and love of Belle, his companion dog. In Memoriam of John P. Levy: scientist, molecular biologist, colleague, and friend an unsung hero of medicine: constructing safe and effective gene-therapy vectors for the next generations.
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