Biotech Lecture 1

Molecular biology (definition)

The study of biology on a molecular level, including the structure, function, and makeup of biologically important molecules such as DNA, RNA, and proteins

Recombinant DNA technology

A set of techniques for manipulating DNA: identification and cloning of genes; study of expression of cloned genes; production of large quantities of gene product

Genetic engineering

The process of transferring DNA from one organism into another that results in a genetic modification

Biotechnology (definition)

Production of goods and services using biological organisms, systems, and processes

Disciplines contributing to molecular biotechnology

Molecular biology, microbiology, biochemistry, immunology, genetics, chemical engineering, cell biology

Products of molecular biotechnology

Crops, drugs, vaccines, diagnostics, livestock

500 B.C.E. biotech milestone

First "antibiotic": moldy soybean curds (tofu) used to treat boils (China)

100 C.E. biotech milestone

First "insecticide": powdered chrysanthemums (China)

Variolation (1720s-1780s)

"Immunizing" against smallpox by infecting patients with material from pustules of patients with a mild form of the disease (variola minor)

1797: first vaccination

Edward Jenner took pus from a cowpox lesion and inserted it into an incision on a boy's arm to prevent smallpox

1830

Proteins are discovered

1833

First enzyme is discovered and isolated

1857: Louis Pasteur

Proposed that microbes cause fermentation; later experiments supported the germ theory of disease

1865: Gregor Mendel

Discovered the laws of inheritance by studying flowers (peas) in his garden; the science of genetics begins

1928: Alexander Fleming

Discovered the antibiotic penicillin by chance when he realized Penicillium mold kills bacteria

Fleming's Nobel Prize (1945)

Shared with Ernst Boris Chain and Howard Walter Florey

1944: Avery, MacLeod, McCarty

Proved that DNA carries genetic information

Avery-MacLeod-McCarty experiment result

Adding S-strain DNA to R-strain bacteria killed the mice; adding S-strain protein did not

1953: Watson and Crick

Described the double helical structure of DNA; shared the 1962 Nobel Prize in Medicine or Physiology with Maurice Wilkins

1955: Frederick Sanger (protein)

Determined the amino acid sequence of insulin; won the 1958 Nobel Prize in Chemistry for work on protein structure, especially insulin

1982: Humulin

Human insulin produced in genetically modified bacteria; the first biotech drug approved by the FDA

1966: genetic code

The genetic code for DNA was determined (Nirenberg, Holley, Khorana shared the 1968 Nobel Prize in Physiology or Medicine)

Genetic code basics

Three bases (a codon) code for one amino acid

1970: Hamilton Smith

Isolated the first site-specific restriction enzyme (HindII); shared the 1978 Nobel Prize with Werner Arber and Daniel Nathans

Restriction enzymes

Site-specific endonucleases that cut DNA into reproducible pieces; pieces can be linked to bacterial vectors and put into bacterial hosts (start of gene cloning and recombinant DNA technology)

1973: Cohen and Boyer

Perfected genetic engineering techniques to cut and paste DNA using restriction enzymes

1977

First expression of a human gene in bacteria

1975: Kohler and Milstein

Developed technology to produce monoclonal antibodies; shared the 1984 Nobel Prize with Niels Jerne

Monoclonal antibodies

Highly specific, purified antibodies derived from only one clone of cells that recognize only one antigen

Monoclonal antibody production (hybridoma)

An antibody-producing spleen cell fuses with a myeloma cell to make a hybridoma cell; a single hybridoma clone yields one specific antibody

Monoclonal antibody drug examples

Humira and Keytruda (foundation of the modern biologics industry)

1975-1977: DNA sequencing

Sanger and colleagues, and Maxam and Gilbert, developed rapid DNA sequencing methods

1980 Nobel Prize in Chemistry

Half to Paul Berg (recombinant DNA); half to Walter Gilbert and Frederick Sanger (determining base sequences in nucleic acids)

1978-1981: Joachim Messing

Rutgers pharmacist and PhD; developed the shotgun DNA sequencing method

Uses of cloning, shotgun sequencing, site-directed mutagenesis

Sequence large DNA molecules like human chromosomes and engineer genes and proteins

1981: transgenic animals

First transgenic animals produced by transferring genes from other animals into mice

1981: first GMO patent

Patent granted for bacteria that can break down crude oil

1983: PCR

Polymerase chain reaction conceived; makes unlimited copies of genes and gene fragments

Kary Mullis

Won the 1993 Nobel Prize in Chemistry for PCR

1986: first recombinant vaccine

Hepatitis B vaccine

1986: first biotech anti-cancer drug

Interferon

1990

Human Genome Project launched (international effort to map all genes in the human genome)

2001

Draft version of the human genome published

2022

First complete, gapless telomere-to-telomere human genome sequence reported

2005: first GLP-1 agonist

Exenatide (Byetta), approved by FDA for type 2 diabetes

Exenatide structure

A 39-amino-acid peptide

GLP-1 timeline: 2010

Liraglutide (Victoza): first once-daily GLP-1 receptor agonist

GLP-1 timeline: 2014

Dulaglutide (Trulicity): once-weekly dosing

GLP-1 timeline: 2017

Semaglutide (Ozempic): once-weekly dosing

GLP-1 timeline: 2022

Tirzepatide (Mounjaro): dual GIP and GLP-1 agonist

GLP-1 timeline: 2026

Oral semaglutide (Wegovy): first oral GLP-1 agonist approved

2006: HPV vaccine

Recombinant vaccine against human papillomavirus (Gardasil) approved; HPV causes genital warts and can cause cervical cancer

2012: CRISPR-Cas9

Genome editing; 2020 Nobel Prize in Chemistry to Emmanuelle Charpentier and Jennifer Doudna

Casgevy (exa-cel)

First CRISPR-based gene-editing therapy; FDA approved Dec 8, 2023 for sickle cell disease (age 12+ with recurrent vaso-occlusive crises)

Casgevy mechanism

Patient's own hematopoietic stem cells are edited ex vivo with CRISPR-Cas9 to reactivate fetal hemoglobin (HbF), then reinfused after conditioning

Lyfgenia (lovotibeglogene autotemcel)

Lentiviral gene therapy for sickle cell disease, approved the same day as Casgevy (Dec 2023)

CRISPR timeline significance

Roughly a decade from the 2012 discovery (2020 Nobel) to an approved medicine

2014: anti-PD-1

FDA approved anti-PD-1 checkpoint inhibitors

PD-1 inhibitor mechanism

Blocks PD-1 on T cells (PD-1/PD-L1 interaction with tumor cells) so the T cell can attack the tumor

2017: first CAR-T therapy

Kymriah (tisagenlecleucel, CTL019), anti-CD19 CAR T cells

CAR-T process

Leukopheresis to collect patient T cells → retroviral transduction with anti-CD19 CAR → preconditioning chemotherapy → infusion of CAR T cells

2020: mRNA vaccines

SARS-CoV-2 (COVID-19) mRNA vaccines; mRNA encodes the spike protein, made by the cell's ribosomes

2024: afami-cel (Tecelra)

FDA-approved engineered T-cell therapy for advanced (metastatic) synovial sarcoma

Afami-cel mechanism

Patient's T cells are engineered with a T-cell receptor that better recognizes and binds MAGE-A4 on cancer cells

2026: Rasonque (daraxonrasib)

FDA approved Aug 26, 2026; once-daily oral RAS(ON) inhibitor for metastatic pancreatic adenocarcinoma after at least one prior systemic therapy

Daraxonrasib mechanism ("molecular glue")

Binds cyclophilin A; the complex clamps active GTP-bound RAS, inhibiting mutant KRAS, NRAS, and HRAS (not just one allele like G12C)

RASolute 302 trial result

Median overall survival 13.2 vs 6.7 months vs chemotherapy (HR 0.40); response rate 30% vs 11%; no companion diagnostic required

Significance of daraxonrasib

RAS was judged undruggable for four decades; first medicine to inhibit it broadly

Central dogma

DNA → RNA → protein (replication, transcription, translation)

Transcription (lecture)

Information coded in the base-pair sequence of DNA is passed to RNA

Translation (lecture)

Information in RNA is passed to proteins; it never passes from proteins to nucleic acids

Human protein-coding gene count

About 20,687 genes (only about 1,796 more than worms; fewer than corn, rice, or wheat)

Human vs cereal difference (Mukherjee quote)

Not gene number but the sophistication of gene networks: "It is not what we have; it is how we use it"

1 base

1 nucleotide = 1 basepair (bp)

Genome size units

1000 bases = 1 kilobase (Kb); 1000 kb = 1 megabase (Mb); 1000 Mb = 1 gigabase (Gb)

Polymorphism

An area of DNA sequence that varies from person to person

SNP

Single nucleotide polymorphism: a single base differs from the usual base at that position

CNV

Copy number variant: the number of repeats of a DNA sequence at a location varies between people

Indel

A DNA sequence is present (insertion) or absent (deletion) at a location, varying between people

How modern drug discovery differs

Begins with understanding the molecular basis of a disease (not just symptoms); uses disease models and genetics to pinpoint drug targets and therapeutics

Druggable genome

The subset of genes whose protein products can bind small, drug-like molecules (estimated ~4,500 genes; Hopkins & Groom 2002)

Which biomolecules are drug targets?

Mainly proteins with binding sites (not polysaccharides or lipids)

Drug target counts

1,578 FDA-approved drugs act through 893 biomolecules; only 667 are human proteins

Four privileged target families

GPCRs, ion channels, kinases, nuclear receptors (about 70% of human targets)

Tdark vs Tclin

Tdark = understudied proteins (NIH Illuminating the Druggable Genome); Tclin = proteins through which an approved drug acts (~704)

Druggable (lecture's lesson)

Describes what chemistry has learned to reach, not a fixed property of a gene

Rational drug design

Combining a specific molecular target with detailed knowledge of intermolecular binding interactions can dramatically accelerate drug invention

Small molecule drug: properties

MW <1000 (preferably <500); structure and purity determined; API usually a solid; often a solid dosage form (tablet, capsule); often dosed orally

Small molecule example

Atorvastatin (Lipitor)

Small molecule advantages

Oral bioavailability; wide distribution (reach intracellular and CNS targets); easier manufacturing/formulation; multiple dosage forms; low-cost generics after patent expiration

Sovaldi

Gilead hepatitis C drug (~$90K per patient) that cures the disease

Biologics: properties

High MW (>>1000); primary structure defined but post-translational modifications less so; not orally deliverable; generally don't penetrate cells; formulation stability challenging; parenteral (s.c., i.v.); hard to generericize (biosimilars)

Biologic example

Humira (bound to TNF-alpha)

Biologics advantages

Resistant to generics; higher prices tolerated; ~20% annual growth; high target selectivity; no CYP450 or hERG ion channel interactions; can hit targets small molecules can't (protein-protein interactions); toxicity generally mechanism-based ("superpharmacology")

Top-selling biologics (examples)

Humira, Enbrel, Remicade, Neulasta, Rituxan, Lantus, Avastin, Epogen

Drug discovery funnel stages

High-throughput screening → hit to lead → lead optimization → candidate seeking → preclinical development → Phase I → Phase II → Phase III

High-throughput screening

Thousands of compounds; IC50 determination and hit triage

Hit to lead

Hundreds of compounds; selectivity assays, in vitro efficacy, tier I ADME/physical chemistry

Lead optimization

Dozens of compounds; in vivo efficacy (preclinical proof of concept), tier II ADME

Candidate seeking

1-3 compounds; second-species PK, PK/PD modeling, salt and crystal form selection