bme final notes

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BME Final Notes

December 16, 2014

1 Lecture 25 - Gene Therapy

Facts:

• First proposed in 1970’s

• First gene therapy attempts to treat immune deficiency in 1990’s

• Student dies in 1999 at Penn from a massive immune response relatedto a trial with adenoviral vectors to treat X-linked liver disease

• Gene therapy success in treating retinal disease in 2007

• Gene therpay for red-green color blindness in adult primates

1.1 A comeback for gene therapy

Gene therapy targeted: Adrenoleukodystrophy (ALD)

• group of genetic disorders called leukodsytrophies that cause damageto the myelin sheat, which is an insulating membrane that surroundsnerve cells in the brain

• people with ALD accumulated high levels of saturaed, very long chain

fatty acids (VLCFA) in the brain and adrenal cortex because theydon’t produce the enzyme that breaks down these fatty acids normally

• Loss of myelin and progressive dysfunction of the adrenal gland areprimary characteristics of ALD

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• Common subtype of the disease is the X-linked form, involves abnor-

mal gene located on the X-chromosome. Women are carriers, menmore severely affect3ed

• Onset of X-ALD can occur in childhood or in adulthead, most severeis childhood with onset between ages 4 and 10

• most common symptoms are behavioral changes such as abnormalwithdrawal or agression, poor memory, poor school performance

• OTher symptoms: visual loss, learning disabilities, seizures, poor speecharticulation, difficulty swalling, progressive dimentia.

• prognosis is poor because of progressive neurolgiical deterioration

• Death occurs within 1 to 10 years after symptom onset

Gene therapy approach:

• Whose cells were modified?

• What/how cells were modified

• how were corrected cells delivered

• Did boys undergo any treatment before the cell/gene delivery

• how long were boys monitored after therapy

• which cells contained corrected gene, which percentage of cells havecorrected gene?

Why are hematopoietic stem cells (HSCs) used for gene therapy or trans-plantation to treat ALD?

Myeloid cells:

• Myeloid Cells in CNS are predominatly yolk sac- derived from earlyembryonic days

• in neurodgenerative diseases, bone marrow-derived phagocytes can en-graft in the brain

• these myeloid cells originate from ciruclating Ly6Chi monocytes orfrom bone marrow derived progenitors

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More questions:

• Did the lentiviral delivery method rely on a gene-targeted approach(example integration into a specific gene locus or was it based on(more-or-less) random integration into the genome?Answer: Lentiviral vector integration occurs at random sites. Appear-ance of clonal cells can indicate positive selection of clones with in-creased growth potential caused by vector insertion into critical genes.Such insertions could then lead to cancer.

By contrast, polyclonal hematopoietic cells suggest random insertioninto many different HSCs without affecting growth characteristiics of these cells

• ”Highly polyclonal hematopoietic cells transduced with the ABCD1gene, which is maintained throughout the follow-up time without ev-idence for sustained expansion of individual clones or enrichment of common integration sites.” What does this mean?

• What’s the advantage of gene therapy with autologous cells comparedto allogeneic transplantation? Think of donor availability treatmentof donor and recipient, risks of the procedure

• What is the relevance of the statmeent that granulocytes, monocytes,

T and B cells expressed the corrected protein for 30 months and more,and that lymphoid and myeloid cells contained the vector?

• Why was expression of the corrected gene in only 9 to 14

Why has gene therapy not become an effective treatment for geneticdisease?:

– Short nature of gene therapy. therpaeutic DNA introduced intotarget cells must remain functional and the cells containing thetherapeutic dNA must be long-lived and stable

– Immune response. Risk of stimulating the immune system in away that reduces gene therapy effectiveenss is always a potentialrisk.

– Problems with viral vectors. Viruses are the carrier of choice inmost gene therapy studies. They present a variety of potential

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problems for the ppatient: toxiicyt, immune and inflammatory

responses, gene control, targeting issues. Apparantly there isalso the ”fear’ that the viral vector, once iti is inside the patient,may be able to recover its ability to cause disease

– Multigene disorders. Arise from mutations in a single gene arethe best candidates for gene therapy. Unfortuantely, some of the most commonly occuring disoders such as heart disease, highblood pressure, Alzehimers, arthritis, diabetes, are caused by thecombined effects of variations in many diseases.

– Ethical questions to consider

∗ what is normal and what is a disability disorder, who decides?

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are disabilities diseases and do they need to be cured?∗ Does searching for a cure demean the lives of individuals

presently affected by disabilities?

∗ Somatic Gene therapy (done in adult cells of persons knownto have the disease) versus egg and sperm cell (germline genetherapy)

∗ Gene therapy very expensive. Who will use have access andpay for it

Gene insertion can disrupt other genes and cause increased proliferation.Why? A random insertion of a corrected gene can cause enrichment of theclonal population.

2 Lecture 26: Cancer Therapies: Kinase Inhibitors

and Protein Engineering

Questions:

• is Gleevec the final solution?

Chromic Myeloid Leukemia (CML) and bcr-abl gene

• Abnormal chromosome develops Chromosomes in blood cells swapsection with each other. A section of chromosome 9 switches place witha section of chromosome 22 (Philadelphia chromosome), creating anextra-short chrom. 22 and extra long chromosome 9.

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• Abnormal chromsome creates a new gene Philly chromsome cre-

ates a new gene. Gense from C9 combine with genese from C22 tocreate BCR-ABl. BCR-ABL gene contains instructions that tell theabnormal blood cell to produce too much typrosine kinase (protein).Tyrosine kinase promotes cancer by allowing certain blood cells togrow out of control.

• New gene allows too many diseased blood cells Blood cells orig-inate in bone marrow. When it functions normally, it produces imma-ture stem cells (blood stem cells) in a controlled way. These cells thenmature and specialize into various types of blood cells which circulatethroughout your body (red/white cells, platelets). This doesn’t workwell in CML. Tyrosine kinase causes too many white blood cells with

abnormal Philly chromsome. These diseased white blood cells don’tgrow and die normally, they build up in huge numbers and crowd outhealthy cells and damage bone marrow.

Gleevec is an ATP competitive inhibiitor. binds to cleft between N andC lobes of the kinase domain of BCR-ABL. Gleevec (Imatinib).

Antibodies as therapies:

• Many breast cancer cells overxpress human epithelial growth factorreceptors (HER), which are part of the receptor tyrosine kinases family

• HER family receptors must dimerize for activation

• specific antibodies that bind to HER2 can prevent its dimerization.Herceptin is an antibody used to treat many breast cancers. Herceptin

– Herceptin - monoclonal antibody, targeted cancer therapy

– produced in lab by making mulltiple copies of a single cell

– Works in 2 ways: blocks HER2 kinase activity, signals and facil-itates immunglobulin to destroy cancer cells

– designed to recognize the HER2 located on the cell membrane,

which is overexpressed in cancer cels– when Herceptin is present, it binds to extracellular domain of

HER2 receptor and prevents HER2 from forming homo/heterodimerswith any other HER family members.

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– This prevents several intracellular signaling cascades that can

drive carcinogenesis– At the same time, immunlgobulin will detect the cancer cell

bound by Herceptin antibodies

– cancer cell is surrounded by immune cells and brought to degre-dation

• Another approach to block HER signaling: Bivalent ligands to BiasErbB receptor mediated signaling and phenotypes:

– ErbB receptor family is dysregulated in many cancers

– its therapeutic manipulation by targeted antibodies and kinase

inhibitors has resulted in effective chemotherapies– A new approach that directs ErbB receptor interactions, resulting

in biased signaling and phenotypes.

– ErbB receptors are necessary to dimerize to signal, bivalent

3 Biotech Lecture

Mission: Catalyze and promote research, eduaction and training in the lifesciences with advanced technologiesCEnter includes: fee-for-service research, tech testing and dev

4 Practice Final 2013

4.1 Problem 1

Gene therapy: ”Comeback for Gene Therapy” for treating adrenoleukodys-trophy (ALD)

4.1.1 Part A

The team modified hematopoietic stem cells from patients with a lentiviralconstruct in vitro and then reintroduced the cells with the correct gene

expression back into the patient.

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4.1.2 Part B

The researchers delivered a corrected version of the ABCD1 gene into thecells. It was randomly inserted into the genome. Interestingly (for me), thechances for random genomic integration are higher than in targeted genecorrection by homolgous recombination. This is apparently rare, it wouldrequire a separate selection step to identify successfully modfied cells. Ran-dom integration has a risk associated with it in that insertion could disruptendogenous gense and result in uncontrolled proliferation. Researchers how-ever found no enrichment of individual clones

4.1.3 Part C

Question: Assume you want to deevelop a gene therapy for muscular dys-trophy using a viral construct that inserts a corret version of the dystrophingene into patients’ cells. Your colleague suggested using a local in viv de-livery strategy to modfiy muscle cells and adult stem cells inside the muscletissue, for example by injecting viral particles into the muscle. Is this rea-sonable?It would be challenging to modify all of the affected muscles throughout thebody using a local delivery approach. This would require getting the virusinto each muscle. Better Approach: systemic delivery. However, modifyingstem cells and re-introducing them to the patient also has several limita-tion, since getting stem cells to infiltrate all muscles is also difficult. In vivodelivery of viral particles can trigger an immune response.

4.2 The immune system and vaccinations

Humoral Immunity: Antibodies produced by the bodyCelullar immunity: cytotxic T-lymphocytes (CTL)

4.3 Early Vaccines vs subunit vaccines

Early vaccines were empirically derived vaccines that were based on atten-uated pathogens, which is either a weakened form of the original pathogenor a related, less toxic organism (cow pox instead of human pox.Subunits vaccines are based on subunits, or surface proteins of the pathogenthat are used to ”train” the immune system, without the risk of an actualinfection occurring.

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4.4 AntiCancer Therapies

4.4.1 Part A

What’s the principle of Gleevec?Competitive inhibitation for Bcr-abl kinase, which is a hyperactive kinase re-sulting from chromosomal translocation (Philadelphia chromosome). Gleevecis very highly specific to the Bcr-abl kinase, which means it has little effecton normal cells which lack this fusion protein.

4.4.2 Part B

What is Herceptin? In contrast to Gleevec, what is the working principle?Many cancers massively overexpress HER. Herceptin is an antibody that

binds to HER2 and prevents its dimerization, preventing downstream sig-nalling. Herceptin bound to HER2 on surface of cancer cells also mobilizesimmune system, targeting the cancer cells for degradation

4.4.3 Part C

In Jay et al, researchers used a bivalent ligand consisting of two neuregulindomains to induce dimerization of HER3. What is special about HER3 vs.other HER family members? Would the approach work for HER4?

HER3 contains a neuregulin receptor domain on the cell surface, but

doesn’t have a cytoplasmic kinase domain. It can only signal when dimeriz-ing with other HER family members. By sequestering HER3 in homodimers,it isn’t available to form heterodimers iwth other HER family members. Thisapproach won’t work for cells expressing HER4, which aso binds to neureg-ulin, since the bivalent protein would also induce dimerization of HER4 andactivate its downstream signalling.

4.5 Question 4: Microfluidic devices

Continuous flow microfluidic PCR can complete a 30 cycle PCR reactionmuch faster than convential well based PCR machine because standard PCRmachines need to heat up and cool down the entire sample block/chamber,

which has large ”thermal mass.” In continuous flow PCR, only sample needsto warm as it passes through different temperature zones, surface to volumeratio is larger and the sample volume smaller in MF devices.

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OtherShit:

Increase residence time in microfluidic segment is to increase cross-sectionalarea, reduce average velocity

Reynolds number in microfluidic systems is usaully very small (<< 1),Flow is laminar. Mixing of adjacent liquids occurs only by diffusion andtherefore takes a long time. Microfluidic devices must havea long segmentto provide sufficient residence time for reagants to mix.

4.6 Various short Answer Questions

4.6.1 DNA/RNA direction synthesis

5 to 3 prime direction, new ribonucleotides can only be added to the -OHgroup at the 3’ position

4.6.2 Size in Nucleuotides for typical primer

Primer length is usaully 18-22 in length. FOr real-time PCR, melting Tshould be 58-60 degrees. Optimal extension temperature for PCR reactionswith Taq polymerase is 72 degrees.

4.6.3 Genomic Organization and Transcription

• Eukaryotes have introns and exons

• Prokarytoes have operons (clusters of related genes for specific func-tion)

• In prokaryotic cells, transcription and translation can occur in paralleland in the cytoplsm

• In prokaryotes, trnaslation can occur in multiple sites of RNA at sametime

4.6.4 3 essential elements to cloning and epxression vectors

Need multipling cloning site (polylinker), selection marker (eg. antibiotic),

origin of replication

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4.6.5 Rest of the Short answer

• Isopycnic centrifugation is independednt of time at long times

• Tandem mass spectrscopy typically uses elecrospray (electrospray ion-ization is a soft ionization that keeps protein intact) to ionize proteinsand peptides of interest in the first stage to select ou specific mass tocharge ratio that can then be selected in the second stage (fragmentedby collision with gas particles and the analyzed to determine proteinidenty)

• MALDI stends for matrix assisted laser desorption ionization, is anionization technique

• current cost to sequence an individual’s entire human today costsabout 5000 dollars

• RPKM - reads per kilobase per million mapped reads. this obtains theexpression levels normalized for gene length. Number of total mappedreads (account for differences in amount of sample and sequencingefficiency b/w different samples)

• Housekeeping gene and endogenous control. You don’t need to know itwith RNA-sequencing because the normalization is performed basedon the number of total reads, and this accounts for the differencesin the amount of RNA in each sample and variations in sequencing

efficiency. Normalizing by the length of the gene further accounts forthe increased chance of finding partila transcripts from longer genes

5 Practice Final 2012

5.1 Question 1: Gene Therapy

5.1.1 Advantages of this gene therapy approach

5.1.2 Why where HSC’s modified

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