Anthony Joseph Raffo, MSc, PhD

SUMMARY

Cellular and molecular biologist with over 20 years in academic and industry research and development. Expertise and experience in molecular biology/biochemistry and clinical/medical research technologies with an emphasis on oncology, virology, inflammation, and immunology. Career has focused on novel experimental therapies and drug discovery and evaluation. Solid expertise in bioassay (especially genomic based) design, development, and validation.

Many years of experience in human and animal models cell and tissue acquisition, cataloging, storing and experimental utilization. Trained on BSH-2 and BSH-3 hoods with potentially and very virulent tissue samples under strict aseptic conditions.

Managed and maintained laboratory, tissue culture and vivarium facilities and trained incoming laboratory personnel on the use, treatment and safety issues involved in laboratory operations.

I was also an integral component of grant writing, data presentation, SOP generation and literature research in basic medical terminology and writings. Gave several presentations of my work to international conferences and have received awards for my work.

SKILLS

More than 20+ years bench experience

Aseptic techniques

Manual and automated liquid handling

Microbiology

Biochemical

Biomedical

Cell culture

Mammalian cell culture

Primary human cell culture

Human specimen cataloging, processing for diagnostic assays and storage

Human virus applications

Microscopy, simple, confocal, IHC

ELISA

Chromatography

HPLC

Immunohistochemistry

Confocal microscopy

Molecular biology

Statistical analysis

Western & Northern blots

CGMP

Aseptic technique

Laboratory maintenance

GLP

Clinical research

Data analysis

Laboratory management

Physiology knowledge

Genetic engineering

Transfection

Writing skills

GCP

Spectroscopy

BSL-2 & 3 operations

Maintenance

Microsoft Office applications

Research & Development

SAS

Tutoring & training

Clinical trials

Microsoft Excel

Data collection

Medical writing and terminology

Grant and protocol writing and conference presentations

In vivo expertise

Sanger & Next Generation Sequencing

Flow cytometry and analysis

Data evaluation and analysis

Manuscript preparation and presentation

FACS analyses

Correspondence with clients, colleagues

Manuscript preparation

Assay troubleshooting

Equipment and laboratory management

OVERVIEW

Designed, developed, conducted, and validated numerous experiments/assays using multiple cellular and molecular advanced technologies, either using in vivo clinical animal models and/or ex vivo, in vitro models of human diseases

Independent investigator with a strong belief in internal team and external investigator collaboration to share ideas and expertise

Extensive experience with cell culture techniques, including primary human cells, bio-banking and cataloging, culturing of established cell lines from human, mouse, insect, and bacteria.

Strong molecular biology skills including DNA sub cloning, cDNA library building, DNA mutagenesis design and PCR/RT-PCR techniques (bioinformatics-based primer design and specific application validation)

Cellular, biochemical, and molecular biology assay development and validation, including protein-protein binding assays, protein expression, purification and characterization, ELISA, dot-blot, and Western blot. Biomedical analytical experience and specific bio marker identification and expression analysis via protein detection and quantitation (flow cytometry and IHC image analysis and Western blot quantitation/validation

Hands-on experience with phage/yeast antibody display libraries, designing and screening strategies for the selection of desirable antibodies/peptides against cell surface and secreted biomolecules, Next Generation Sequencing

Biochemical and analytical measurements, experimental design and execution, drug discovery, development and assay design, measurement and validation, molecular and cellular biology assay design, cell line development, small animal handling and husbandry, genomic mutation detection (SNP diagnostics)and engineering with mutation generation plasmid design and isolation with introduction into mammalian, insect and bacterial cells, PCR, qRt-PCR measurement of gene expression, protein, RNA, DNA isolation and examination, Microsoft Office, Word, Excel, Access, PowerPoint for manuscript and grant writing, presentation, lab and personnel management, administrative and team focus experience, electronic lab notebook and record management software, confocal microscopy computer operation record keeping/analysis, qRT-PCR computer operation and analysis, GWAS operation, recording and analysis software, manual and automation liquid handling, in vivo experiment design and manipulation, personnel management and training

Significantly contributed to medical grant and procedural writing and data analysis and presentation

EDUCATION

University of California at Riverside, Riverside, CA

Doctorate in Plant Pathology, GPA: 3.42 September 1987 to June 1991

University of California, Riverside, CA

Master of Science in Molecular Biology and Biochemistry, GPA: 3.75 September 1983 to June 1987

PROFESSIONAL EXPERIENCE

Corium Diagnostics, Norwalk, CT

Genomic Technologist and Safety Officer (Part time) February 1, 2022 to April 15, 2022

COVID-19 saliva diagnostic assay development and validation

Registration and validation of assay and instrument with Yale University School of Medicine

Progressive Diagnostics, Branford, CT

Genomic Technologist and Night Supervisor (Contract) March 18, 2021 to May 1, 2021

COVID-19 assay development and implementation

CoPath Laboratory, Yale University, Pathology Department, New Haven, CT

Genomic Technologist (Per diem temporary) January 11, 2021 to March 6, 2021

Cataloged, processed human nasal swabs and human saliva for the presence of SARS-COV2 (COVID-19) virus and prepared positive specimens for viral sequence analysis

Genomics Laboratory, Sema4 Therapeutics, Branford, CT

Genomics Technologist (Contract) May 20, 2020 to June 22, 2020

Helped develop SARS-COV2 (COVID-19) high throughput automated genomic testing

Human swab samples were collected, cataloged, and processed. Human nasal swab samples were assayed for COVID-19 virus by viral genomic isolation and RT-PCR amplification detection.

All procedures were done with high throughput liquid handling in multi-well plates (96 and 384 well formats)

Initial procedure was to remove disposable nasal swab from an aqueous salt solution and prepare samples for aliquot sampling, then subsequent initial procedure was for genomic RNA isolation and subsequent reverse transcribe to DNA. Both done by high throughput reactions and liquid handling (96 well format)

These plates were then sealed and transported to a nearby facility where high throughput PCR would amplify any viral signals for detection, identification, and analysis

All operations were conducted in BSL-2 hoods with extensive PPE protection

From late November 2017 to mid-2020, I did not pursue scientific positions and pursued my other passions, such as food management and preparation and retail marketing

Department of Neurosurgery, Einstein School of Medicine, Medical Center, Bronx, NY

Associate Research Specialist (Temporary) June 2017 to November 2017

Involved in research to determine the molecular biology and biochemistry of alterations in the glycobiology of abnormal modifications in post translational organization of membrane glycoproteins specifically how Tn-antigen, an oncology biomarker, is increased in brain tissue and how this correlated to the onset of Alzheimer’s disease

Major aim was to determine the activity, and expression, protein and mRNA of key enzymes involved in the synthesis and degradation of glycoprotein sugars and the core Tn-antigen.

A secondary project was to develop the potential of small compounds synthesized within the lab as anti-cancer therapies, specifically, the primary aim of this project was to explore the toxicity of several of these compounds to determine an appropriate dose to destroy brain cancer cells while not effecting hepatocyte function

Department of Immunology and Respiration, Inflammatory Bowel Disease, Research and Development, Boehringer-Ingelheim, Inc, Ridgefield, CT

In Vivo Scientist (Contract) May 2014 to June 2016

Support Scientist to determine molecular mechanisms of chronic bowel inflammation and fibrosis in rodent and human model systems and determine safety and efficacy of experimental BI therapy reagents, both chemical and biological

Major role was in in vivo mouse experimental setup and harvesting of various tissues and in determining the gene expression analysis as effected and modulated by experimental therapy reagents in animal experimental model that reflect human disease

Just prior to my contract expiration, I was developing nucleic acid-based microbiome assays to determine the effect of bowel disease induction and subsequent test therapy introduction on the bacterial content and type isolated from mice fecal samples.

Two years of experience in high throughput RNA isolation from in vivo tissue samples

Clinical Development Laboratory-Genomics, Merck & Co, Rahway, NJ

Biochemist III/Genomics Staff Scientist (Contract) March 2012 to April 2013

Lead Scientist in the preparation and establishment of nucleic acid-based assays, procedures, and protocols, adhering to GLP compliant practices, prior to regulatory approval, used in the clinical trials of potential Merck generated therapeutics for various human disease applications. These responsibilities required detailed medical procedural wring including detailed medical terminology presentations.

·Designed, optimized, and validated a genotyping assay for two potentially key, though poorly defined by standard techniques, single nucleotide polymorphisms (SNPs) in a gene, whose protein is implicated as causal in coronary arterial disease (CAD).

Validation results:

A) Reproducibility - same sample assayed in duplicates, triplicates and/or quadruplicates; 100% for both SNPs

B) Reliability - different samples taken from the same donor, material isolated independently, and analysis completed in independent different runs: 100% for both SNPs

C) Efficiency - ability to positive identify genotype in over 350 different samples: 100% for one SNP and 99.7% in second SNP

D) Sensitivity - both showed the same ability to positively detect each SNP in serial dilutions of starting sample from 30ng/ul to 0.002ng/ul using single donor blood sample treated and isolated by two different protocols

E) Variability - both SNPs were positively identified in 6 donor samples regardless of the isolation protocols used

·Established two next generation sequencing (NGS) platforms at Merck, CDL-G Rahway facility, with the installation of Illumina and Ion Torrent instruments and was the scientific lead on a project to outsource the use of the SMART instrument technology of Pacific BioSciences, Inc.

·Established criteria to test and validate several vendors, which Merck needs as outsourcing partners in patient gene expression analysis. Approved 2 out of 5 vendors and this validation design was used to test additional global Merck outsourcing vendors

·Designed and optimized a FFPE sample core needle biopsy equivalent nucleic acid-based gene expression assay to validate siRNA treatment protocols in human liver cancer samples.

Several of these projects, as well as others, though still in early development, were developed with close interaction with the Merck Biomarkers group at its Kenilworth facility. Our goal was to coordinate biomarker discovery toward the early development of a clinical nucleic acid-based assay for a therapy treatment decision in breast and ovarian cancer. Many of these issues dealt with the smooth communication through EDCMS documentation

Department of Medicine, Columbia University Medical Center, New York, NY

Associate Research Scientist September 1995 to July 2008

Throughout my tenure at CUMC, I was called upon to write research articles, grant proposals, experimental protocols and present laboratory results and conclusions to national and global scientific conferences, most of these felt with pre-clinical and medical terminology and concepts

The Naomi Berrie Diabetes Center

·Established non-evasive imaging of pancreatic beta cells by Positron Emission Tomography (PET) scanning, in an induced rat model or in a spontaneous diabetic prone rat model

·Correlated the imaging data with the appearance of beta cell specific peptides within the serum of these rats as diabetes progresses using mass spectroscopy analysis

·Quantitatively measure the loss of beta cell specific proteins and their mRNAs within the regressed pancreas in these diabetic rats by Western blots, ELISAs and qRT-PCR.

·Imaged and semi-quantitated the localization of several key proteins in the pancreas of these animals and in a rat glucose inducible insulin secreting cell line, by confocal fluorescent immunohistochemistry focusing on pancreatic specific biomarkers

·Molecular bio/biomarker assay development (including flow cytometry and RT-qPCR based). These were basic biomarker validation as markers for type II diabetes progression and/or treatment efficiency issues

·Signal transduction pathways effecting growth, death, differentiation, and senescence, with emphasis on oncology (specifically prostate, breast, melanoma, and lymphoma cancers), immunology and metabolic disorders (diabetes)

Department of Medicine, Division of Medical Oncology, Experimental Therapeutics

·Determined the in situ and in vivo application of a bio-active peptide from the C and N terminus of the key cancer biomarker tumor suppressor protein p53, which can activate some mutant forms of p53 in breast, lung, brain, and prostate cancer cell lines, inducing cell death. These studies included limited DMPK analysis of absorption, metabolism, and kinetics of peptide in cell line studies

·Experience in writing SOPs, protocols, and articles, as well as presenting data and conclusions to large peer scientific audiences with an over whelming desire to learn as much as possible through these interactions and through a conscientious reading of relevant scientific articles and publications

·Designed and constructed bacterial, mammalian and insect vectors for the production and isolation of peptides from several regions of the human DNA repair biomarker protein XRCC-1 Part of this project was to determine the three-dimensional structure of these conserved regions and to correlate this structure with several polymorphisms found in the human population. An additional part of this project was to determine how these polymorphisms affect the DNA repair function

·Therapeutic chemical and biologic agents efficacy studies and combinatorial chemotherapy; DMPK/ADME studies

·Determined the efficacy of certain experimental drugs for the treatment of cancers in situ and in vivo, including toxicology (limited bioanalytical DMPK/ADME in situ studies using HPLC and LC/MS) of these agents either alone or in combination with existing chemotherapy agents. In vivo imaging of patient tumor therapy progression by PET and MRI for prostate cancer patients to evaluate experimental therapy protocols

·In collaboration with Gwen Nichols, MD, a staff Oncologist, I conducted clinical serological and toxicological assays on patient blood samples as part of a clinical study to determine efficacy of chemotherapy regimen in patients with B-cell lymphoma. Cell cycle changes, apoptotic assays, mitochondrial potential, caspase activation and the expression of key apoptotic mRNAs and key biomarker proteins were measured by flow cytometry (FACS), confocal microscopy cellular imaging, Western blot, ELISA, and qRT-PCR gene expression analyses

·Designed and constructed mammalian expression vectors, which expressed micro RNAs, which would interfere and decrease the expression of the anti-apoptotic protein bcl-2. An anti-sense oligomer, which is in clinical trials, has been shown to decrease the viability of human prostate cancer cells, which decreased bcl-2 protein expression by almost 90%. Determined DMPK studies with measurements of absorption, metabolism, and elimination of labeled anti-sense biological oligomers in cell lines. These studies were confirmed using siRNAs, which also dramatically decreased bcl-2 expression. This project suggested that while bcl-2 levels were important in oligomer efficacy, alternative mechanisms, perhaps involving a cellular immune type of response, would also play a key role in anti-sense bcl-2 oligomer action against prostate cancer viability

·As an advisor to a Ph.D. graduate student, I transfected prostate cancer cells with vectors designed to over-express cIAP-1 protein, a key biomarker of chemotherapy resistance. While I isolated clones that expressed only minimal increases in this protein, the cells were resistant to several chemotherapy reagents, However, the growth rates of these cells were substantially lower than mock transfected cells, suggesting a possible role for this protein in cell cycle control (yet to be determined)

·Used siRNA, antisense oligomers, drug, antibody, and peptide therapies

·Gene expression analysis using flow cytometry (FACS), RT-qPCR, qPCR, sequencing, Northerns, run-off transcription, IHC, Westerns, Southerns, FISH, ELIZA, immuno-histochemistry and confocal microscopy

·Small animals (PI on IACUC proposals), xenograph models of cancer, toxicologic assays of chemotherapy agents and IHC imaging studies and drug efficacy models in the development of therapeutic agents

Columbia University Cancer Center

·Setup and organized the micro-array for gene expression analysis core facility of the Institute of Cancer Genetics, Pathology, under its director Professor Riccardo Dalla-Favera, PhD. In this temporary role, I supervised the acquisition of equipment and materials, trained the technician who ran the facility and organized the data management; all in cooperation with Affymetrix application scientists. This facility was designed to determine expression analysis, for pre-clinical and basic research investigations of biomarkers involved in cancer potential and progression.

Department of Urology, Columbia University, New York, NY

Associate Research Scientist September 1992 to September 1995

·Determined the effect of over-expression and gene copy number analysis of the anti-apoptotic biomarker protein bcl-2 (bcl xL) on androgen withdrawal and chemotherapy resistance in human prostate cancer cell lines

·Constructed gene expression vectors that overexpressed either the pro-life bcl-2 or bcl-xl proteins or the pro-apoptosis bax and bak proteins to determine drug sensitivity and chemotherapy resistance molecular mechanisms

·Designed a highly sensitive RT-PCR based assay (chemiluminescence) to detect minimal human prostate cancer cells in the circulation of human prostate cancer patients this assay is used in identifying those patients who would not be a good candidate for the highly evasive radical prostate surgery, because of metastatic circulating prostate cells. This assay targeted the prostate biomarkers PSA and PSM to highlight these circulating prostate cancer cell

·Designed and conducted studies to determine the effect of wild type and mutant p53 over-expression in human prostate cancer cells to hypoxia induced increases in Hif-1 alpha expression and Hif-1 transcriptional activation in the expression of angiogenic proteins controlled by Hif-

Department of Urology, Columbia University, New York, NY

Advisor, Resident Laboratory Research Program September 1994 to September 1995

·Trained and supervised Urology residences on proper animal handling and cell culture techniques.

·Helped residents design and conduct in vivo and in situ experiments in prostate cancer research.

Department of Plant Pathology, University of California, Riverside, CA

PhD Fellow and Post-doctoral Fellow September 1991 to July 1992

·Established diagnostic genomic NA and protein-based assays for several plant RNA viruses.

·Basic research on the molecular mechanisms of RNA viral replication

·Established proof of principle molecular biology methodologies of vector construction based on a simple RNA virus for the expression of external RNA to produce protein products

AWARDS:

American Association for Cancer Research- GlaxoWelcome Investigator Award 2000. Raffo, A.J., Drew, L., Kim, A.L., Brandt-Rauf, P.W., Petrylak, D.P., Do, T. and Fine, R.L. Selective Induction of fas Mediated Apoptosis by p53 Peptide in Human Cancer Cells

American Association for Cancer Research-Intergen Young Investigator Award 1999. Raffo, A.J., Kim, A.L. and Fine, R.L. Formation of nuclear Bax/p53 complexes are associated with apoptotic DNA fragmentation.

American Urological Association. 2003 Moderator of the Poster Discussion Session on Prostate Cancer at the 2003 A.U.A. National Meeting, Chicago

American Association for Cancer Research 1994 Travel Award for Meritorious Abstracts. Raffo, A., Perlman, H., Day, M., Chen, M.W., and Buttyan, R. New anti-apoptosis gene identified in a prostate cancer cell line

American Urological Association. 1995 Best Poster in Disease Staging. De Vries, G., Olsson, C.A., Raffo, A.J., Cam, C., O'Toole, K., Benson, M.C., Buttyan, R. and Katz, A.E. The Molecular staging of prostate cancer using an RT-PCR assay: a study of 100 radical prostatectomy patients.

SCIENTIFIC PUBLICATIONS

Citations for 29 scientific research articles published in peer reviewed scientific journals are available upon request

Select Publications

1) Raffo, A.J., Perlamn, H., Chen, M.W., Day, M.L., Streitman, J.S. and Buttyan, R. Overexpression of bcl-2 protects prostate cancer cells from apoptosis in vitro and confers resistance to androgen-depletion in vivo. Cancer Research 55: pp. 4438-4445, 1995

2) Raffo, A.J., Kim, A.L. and Fine, R.L. The formation of a nuclear p53/Bax complex is associated with chemotherapy induced apoptosis. Oncogene Dec 14;19(54):6216-6228, 2000

3)Raffo, A.J., Lai, J.C., Stein, C.A., Miller, P., Scaringe, S., Khvornova, A. and Benimetsky, L. Antisense RNA downregulation of bcl-2 expression in DU145 prostate cancer cells does not diminish the cytostatic effects of G3139 (Oblimersen). Clinical Cancer Research, May 1, 10(9):3195-3206, 2004

4) Raffo, A., Hancock, K., Polito, T., Xie, Y., Andan, G., Witkowski, P., Hardy, M., Barba, P., Ferrara, C., Maffei, A., Freeby, M., Goland, R., Leibel, R.L., Sweet, I.R., and Harris, P.E.

Role of vesicular monoamine transporter type 2 in rodent insulin secretion and glucose metabolism revealed by its specific antagonist tetrabenazine. J Endocrinol. July 198(1):41-9 2008

5) Kim, A. L., Raffo, A. J., Brandt-Rauf, P. W., Pincus, M. R., Monaco, R., Abarzua, P., and Fine, R. L. Conformational and molecular basis for induction of apoptosis by a p53 C-terminal peptide in human cancer cells. J Biol Chem 274, 349**-*****. 1999

6) Yuehua Mao, Richard Dinnen, Ramon V. Rosal, Anthony Raffo, Patrick

Senatus, Jeffrey N. Bruce, Gwen Nichols, Hsin Wang, Yong-Liang Li, Paul W

Brandt-Rauf, Robert L. Fine

C-Terminal p53 Palindromic Tetrapeptide Restores Full Apoptotic

Function to Mutant p53 Cancer Cells in Vitro and in Vivo

Biomedicines-1739566. 2022. Accepted for publication

PROFESSIONAL REFERENCES

1.Ernest Raymond, PhD, Principal Scientist for Inflammatory Bowel Disease, Department of Immunology and Inflammation, Research and Development, Boehringer-Ingelheim, Inc, 900 Ridgebury Rd, Ridgefield, CT 06811. adsjq1@r.postjobfree.com 203-***-****. Supervisor while at BIP-US-R I&I

2.Matthew Marton, Ph.D., Director of Research Sciences, Clinical Development Laboratories- Genomics, Merck & Co., RY50-1D-134, 126 E Lincoln Ave., Rahway, NJ 07065 adsjq1@r.postjobfree.com 732-***-****. Supervisor at Merck & Co., CDL-Genomics

3.Paul W. Brandt-Rauf. MD, Ph.D., Distinguished Professor and Dean, School of Biomedical Engineering, Science and Health Systems, Drexel University, 3141 Chestnut St., Philadelphia, PA 19104, Bossone 718-A,. adsjq1@r.postjobfree.com 215-***-****. Prior colleague at Columbia

University Medical Center, Dept. of Medicine, Experimental Therapeutics

4.Paul Benya, PhD, Recall Scientist, UCLA Orthopedic Hospital Department of Orthopedic Surgery, David Geffen School of Medicine at UCLA, University of California, Los Angeles, CA

adsjq1@r.postjobfree.com 310-***-****. Prior supervisor while at USC Dept of Orthopedics (prior to graduate education-not included in resume text)

5.Ralph Buttyan, Ph.D., Senior Research Scientist, Vancouver Prostate Centre Professor, Department of Urologic Sciences, University of British Columbia Jack Bell Research Centre adsjq1@r.postjobfree.com 2660 Oak Street, Vancouver, BC V6H 3Z6

Phone: 604-***-****. Supervisor while at Dept of Urology, Columbia University

6.Robert L Fine, MD Oncologist specializing in Internal Medicine, Hematology/Oncology And Medical Oncology, Department of Medical Oncology 171 Ft. Washington Ave, New York, NY 10032 adsjq1@r.postjobfree.com, 212-***-****, prior supervisor during several years at Columbia University Medical Center

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