mbb 304 q4

covers lectures 8-10

CRISPR/Cas9

A bacterial defense mechanism adapted for genome editing, where a guide RNA directs the Cas9 endonuclease to introduce a double-strand break (DSB) at a specific DNA target sequence.

Cas9

An endonuclease protein that acts as molecular scissors to cut both strands of a targeted DNA sequence.

TracrRNA vs. Single Guide RNA (sgRNA)

TracrRNA is a bacterial scaffold RNA that links guide RNA to Cas9; Charpentier and Doudna engineered a simplified single guide RNA (sgRNA) combining both components for reprogrammable gene targeting.

NHEJ vs. HDR Repair

Cells repair CRISPR double-strand breaks via Non-Homologous End Joining (NHEJ, error-prone ligation causing indels) or Homology-Directed Repair (HDR, precise repair using a donor DNA template).

Casgevy (Exa-cel)

The first approved CRISPR/Cas9 therapy for Sickle Cell Anemia and transfusion-dependent beta-thalassemia, which disrupts an erythroid-specific enhancer of the BCL11A gene rather than editing the mutated beta-globin gene directly.

BCL11A Repressor

A zinc finger transcription factor expressed in blood stem cells and brain that represses fetal gamma-globin expression after birth; disrupting its erythroid enhancer reactivates fetal hemoglobin (HbF) synthesis.

Autologous Stem Cell Transplant (Casgevy Workflow)

A therapy where a patient's own blood stem cells are harvested via mobilization and apheresis, edited ex vivo with CRISPR/Cas9, and reinfused after myeloablative conditioning to prevent graft-versus-host disease.

Off-Target Edits and Genomic Risks

Unintended genomic modifications caused by CRISPR/Cas9, including off-target cuts, large deletions, and chromosomal rearrangements that carry a potential risk of blood cancers.

Adenovirus Vector

A large (36 kb) non-integrating viral vector with high immunogenicity, used in vaccines and localized therapies but capable of triggering severe immune responses.

Adeno-Associated Virus (AAV) Vector

A small (4.8 kb) non-integrating viral vector with low immunogenicity and serotype specificity, widely used for in vivo gene delivery.

Retrovirus vs. Lentivirus Vectors

Both are integrating vectors carrying ~8 kb genomes; retroviruses carry a higher risk of insertional mutagenesis and immunogenicity, while lentiviruses have lower mutagenesis risk and are widely used in ex vivo cell therapies.

Jesse Gelsinger Trial (1999)

A landmark gene therapy case where an OTC deficiency patient died from a severe systemic immune reaction to an adenoviral vector, underscoring the critical need for rigorous safety assessment, informed consent disclosures, and managing conflicts of interest.

Nuclear Envelope & Nuclear Lamina

The nuclear envelope is a double membrane structure dotted with nuclear pore complexes; the nuclear lamina is an intermediate filament meshwork beneath the inner membrane providing structural support, genome tethering, transcription factor sequestration, and signaling platforms.

Intermediate Filament Assembly

A structural hierarchy where alpha-helical monomers form coiled-coil dimers, which assemble into anti-parallel tetramers; eight tetramers pack laterally into unit-length filaments that elongate end-to-end.

CAAX Motif

A C-terminal protein motif (Cysteine, Aliphatic, Aliphatic, any amino acid) on prelamin A and Ras proteins that directs a four-step lipidation and proteolysis maturation pathway.

Lamin A 4-Step Maturation Model

Prelamin A maturation sequence:

1. Farnesylation of Cys by FTase

2. Tripeptide (-AAX) cleavage by ZMPSTE24

3. Carboxymethylation of Cys by ICMT

4. Second cleavage of the terminal 15 amino acids by ZMPSTE24 to release mature Lamin A.

Hutchinson-Gilford Progeria Syndrome (HGPS)

An ultra-rare autosomal dominant condition characterized by accelerated aging, growth failure, hair loss, joint stiffness, and premature cardiovascular disease leading to death at ~14.5 years untreated.

LMNA c.1824C>T (p.G608G) Mutation

A de novo "silent" point mutation in exon 11 of the LMNA gene that activates an internal cryptic 5' splice site during precursor mRNA processing.

Cryptic Splice Site & Progerin Production

Aberrant splicing caused by the G608G mutation deletes 150 nucleotides (50 amino acids) from prelamin A mRNA, generating Progerin, which lacks the second ZMPSTE24 cleavage site.

Progerin Pathogenesis & Nuclear Blebbing

Because Progerin retains a permanent farnesylated and methylated C-terminus, it remains anchored to the inner nuclear membrane and acts as a toxic mutant protein that disrupts lamina architecture, causing nuclear blebbing, heterochromatin loss, and DNA damage accumulation.

Zokinvy (Lonafarnib)

An FDA-approved Farnesyltransferase inhibitor (FTi) that blocks the farnesylation step of prelamin A/progerin synthesis to reduce toxic nuclear accumulation in progeria.

Lysosomal Storage Disorders (LSDs)

A group of over 50 inherited metabolic disorders caused by deficiencies in lysosomal acid hydrolases or membrane transporters, leading to the toxic accumulation of undigested macromolecules.

Tay-Sachs Disease (TSD)

An autosomal recessive lysosomal storage disorder caused by mutations in the HEXA gene (encoding the alpha-subunit of beta-hexosaminidase A), leading to GM2 ganglioside accumulation in neuronal cells.

Beta-Hexosaminidase A (Hex A) Isozyme

A dimeric lysosomal enzyme composed of one alpha-subunit (HEXA, chromosome 15) and one beta-subunit (HEXB, chromosome 5) that cleaves N-acetylgalactosamine (GalNAc) from GM2 ganglioside to produce GM3 ganglioside.

GM2 Activator Protein (GM2AP)

A lipid transfer protein that binds GM2 ganglioside in the lysosomal membrane and presents it to Hex A for enzymatic cleavage.

Tay-Sachs Clinical Heterogeneity & HexA Activity

Residual mutant HexA activity determines onset and severity: Infantile TSD (<0.5% activity, death by 4-5 yrs), Juvenile TSD (~5-10% activity), and Adult TSD (~10-20% activity).

Common Tay-Sachs Mutations

A 4-bp insertion in exon 11 (+TATC) accounting for ~80% of carriers in the Ashkenazi Jewish population, and a 7.5 kb deletion of exon 1 prevalent in French Canadian carriers.

Cherry-Red Spot on Retina

A classic ophthalmic diagnostic sign where the fovea appears bright red because retinal ganglion cells surrounding it swell with accumulated GM2 ganglioside and turn opaque pale/white.

Phenylketonuria (PKU)

An autosomal recessive metabolic disorder caused by mutations in the PAH gene, leading to deficient phenylalanine hydroxylase activity and toxic accumulation of dietary L-phenylalanine.

Phenylalanine Hydroxylase (PAH) Mechanism

An enzyme primarily located in liver and kidney cells that converts essential amino acid L-phenylalanine into L-tyrosine using molecular oxygen, iron (Fe2+), and the cofactor tetrahydrobiopterin (BH4).

LAT1 (SLC7A5) Transporter & BBB Competition

An L-type amino acid transporter at the blood-brain barrier; high blood phenylalanine outcompetes tyrosine and tryptophan for LAT1 transport into the brain, causing central tyrosine/tryptophan deficiency and reduced dopamine, norepinephrine, and serotonin synthesis.

PKU Newborn Screening

Mandatory blood spot screening (heel prick) conducted within 24 hours of birth to detect elevated blood phenylalanine levels, enabling early intervention to prevent severe intellectual disability.

Sapropterin (Kuvan)

A synthetic co-enzyme form of tetrahydrobiopterin (BH4) that stimulates residual PAH enzyme activity in responsive PKU patients.

Pegvaliase (Palynziq)

An enzyme substitution therapy consisting of PEGylated recombinant phenylalanine ammonia lyase (rAvPAL) from cyanobacteria (Anabaena variabilis) that breaks down systemic phenylalanine into trans-cinnamic acid and ammonia.

PEGylation Benefits

Attachment of polyethylene glycol (PEG) chains to therapeutic proteins, which increases solubility and stability, decreases accessibility to proteolytic enzymes and antibodies (reduced immunogenicity), and extends blood circulation time.

PKU vs. Tay-Sachs Therapeutic Accessibility

PKU is treatable because its substrate (phenylalanine) comes from diet and accumulates systemically in blood/body fluids outside cells; Tay-Sachs lacks a cure because GM2 ganglioside is synthesized and accumulated endogenously inside neuronal lysosomes, making delivery across the blood-brain barrier and into lysosomes extremely difficult.