Micro 9: Viral Vectors & Gene Therapy

with LPL deficiency patient cannot break down _______; the clinical presentation is (4)

pt cannot break down fats; - abdominal pain -acute and recurrent pancreatitis -eruptive cutaneous xanthoma -hepatosplenomegaly

what is the challenging part of delivery?

remove disease causing components of virus and insert functional clones to treat

what is a disadvantage of murine-based retrovirus vectors?

require replicating cells for genome integration

how do you get CAR into patient's Tcells?

retrovirus, viral vector used to transduce Tcells ex vivo

what is the second step of CAR production?

scFv is fused to extracellular CD8 domain and cytoplasmic CD3

what is Clustered Regularly Interspaced Short Palindromic Repeats CRISPR/Cas9?

segments of bacterial DNA containing short, repetitive base sequences that form hairpin structures

what is the second step in making a retroviral gene therapy vector?

sequences from virus that result in packaging of genetic material into virus (aka packaging signals) are removed, put in plasmid -therapeutic gene of interest is cloned between packaging signals

what is adeno-associated virus (AAV)?

small virus which infects humans and some other primate species

which proteins are deleted for adenoviruses?

structural proteins and proteins that induce cell division

Leber congenital amaurosis cause? result? vector used?

-bialleic mutations in RPE65 gene -blinding retinal diseases -rAAV2 carrying human RPE65 gene

with CAR-T Cell Immunotherapy for Acute Lymphoblastic Leukemia: how are Tcells collected? what are they infected with? how are Tcells reintroduced?

-collected by apheresis -infected by viral vector with CAR -the Tcells are reintroduced via Tcell adoptive transfer

Success with gene therapy for SCID-X1 disease: deficiency? result? what vector was used?

-deficiency of gamma-subunit for IL-2, 4, 7, 9, 15 -causes block T and NK cell differentiation -retrovirus vector encoding gamma-subunit and exvivo CD34+ cells

how does AAV differ from adenovirus? (3)

-does not stimulate inflammation in the host -does not elicit antibodies against self -can integrate on chr19, but inefficiently

first step in making a retroviral vector ends in "packaging cell line"; what is the process?

-genes encoding structural proteins deleted from genome and cloned into plasmid expression vector -plasmid is then transfected into cell; express structural proteins not viral genomes (done in cell culture)

what is involved in controlled gene expression?

-making the correct amount of therapy protein, @ right time -maintain longterm expression

Adenosine deaminase deficiency SCID? deficiency? result? vector used?

-purine metabolism defect -impaired lymphocyte development and function -retrovirus vector containing ADA cDNA and exvivo CD34+ cells

what are the 4 main disease states being targeted for gene therapy intervention?

1 Cancer 2 Monogenic diseases 3 Adenosine deaminase deficiency (SCID) 4 Leber congenital amaurosis

what are the steps involved in development of a genetic therapy?

1 identify a gene responsible for dz; clone functional cDNA 2 delivery 3 control gene expression 4 avoid host immune response

what are 5 factors influencing development of gene therapy??

1 optimize vector safety 2 overcome tech obstacles 3 analysis of human genome 4 expansion of diagnosis industry 5 public acceptance of gene therapy

steps in ex vivo gene therapy treatment of Familial Hypercholesterolemia

1. remove pt liver, treat cells with retrovirus LDL receptor gene 2. discard liver cells that did not take up corrective genes 3. reimplant corrected genes into patient liver

what is the success rate of gene therapy for SCID-X1?

10/11 infants cured

first clinical trial for gene therapy? year, strategy, disorder

1990, ex vivo, Adenosine Deaminase Deficiency

how long after XSCID study did patients begin to develop leukemia?

3 years after treatment

what is Glybera?

AAV1 carrying lipoprotein lipase gene

how is CD19 utilized in treatment?

CAR binds leukemic Bcells with CD19, then activates Tcells to destroy leukemic Bcells

why are retroviruses used to integrate patient's Tcells?

CAR gene will integrate into genome of Tcell

Kymriah specifically recognizes what Bcell marker?

CD19

what is a Cas protein? how does it work with target sequence?

Cas proteins are endonucleases; together with target sequence they recognize and cut exogenous DNA

What is CAR-T Cell Immunotherapy?

Chimeric Antigen Receptor on a patients Tcell; programming patients own cells to attack cancer

what is a Lentivirus? what is the advantage of using it as a vector?

HIV-based gene vector that can integrate into both replicating and non-replicating cells

first step in production of CAR?

In recognition domain, variable region gene segments cloned to construct a single-chain variable fragment

who was the first death directly attributed to gene therapy? what did the patient have? what treatment did the patient receive?

Jesse Gelsinger had ornithine transcrabamylase (OTC) deficiency; he was infused with an adenovirus derived vector directly into hepatic artery

5 types of vectors

adenovirus adeno-associated virus herpes virus liposomes/naked DNA retrovirus

what was the outcome of LCA treatment with rAAV2?

all 12 patients responded well; 6 restored vision, not legally blind

what are most vectors based on?

attenuated or modified versions of viruses

how does an adenovirus enter cell?

binds to specific receptors, delivered to nucleus following uncoating; replicates episomally

what is the success rate of gene therapy for ADA-SCID?

both patients treated in this specific study are home and well

what was the first in vivo gene therapy treating? what did it use?

cystic fibrosis treated with attenuated adenovirus

why is it that liposomes can deliver to many cell types?

do not bind to specific cell receptors

what does the CRSPR/Cas9 system have the capability to do

edit genomic DNA of any species by delivering Cas9 nuclease+ synthetic RNA into cell

why are structural proteins removed when making a vector?

eliminates the possibility that the vector can become a self-replicating virus

what is a retrovirus?

enveloped virus with RNA genome; upon infection of cell converted to dsDNA by enzyme reverse transcriptase

example of cytolytic therapy; what converts prodrug glancyclovir to toxic drug?

expression of thymidine kinase (TK)

how did AAV get its name? why is it advantageous as a gene therapy vector

found in cells simultaneously infected with adenovirus; by itself it is harmless

what accompanies scFV fusion to CD8?

fuse to transmembrane/ cytoplasmic domain of CD3

which type of therapy somatic/germline, is limited to animal models?

germline

what is the goal of delivery?

get genetic material to the appropriate cell types: specific, efficient, safe

first problem with gene therapy that needs to be solved?

how to avoid an immune response; inflammation or antibodies

second problem with gene therapy that needs to be solved?

how to get genes efficiently into non-dividing cells (liver, muscle, neurons)

what is the third problem associated with gene therapy that needs to be solved?

how to get genes integrated so that they will not be deleted or over-expressed

strong evidence leads investigators to believe leukemia is due to "___________ _________________"

insertional oncogenesis

what is germline gene therapy?

involves genetic modification of germ cells; these WILL change the next generation

what does somatic gene therapy involve?

manipulation of gene expression in cells corrective for patient; will NOT be passed on to further generations

what is a vector?

modified, attenuated viruses that are used to deliver payload(gene) into a cell

what was the suggested cause of Jesse Gelsinger's death?

multiorgan failure from anoxia; levels of cytokines suggest systemic inflammatory response syndrome(SIRS)

what is an adenovirus?

non-enveloped virus with large dsDNA genome that accommodates large inserts

what is gene therapy?

novel approach to treating disease based on modifying expression of a person's genes toward therapeutic goal

with correction of ADA-SCID, what did patients undergo before receiving gene therapy?

patients underwent mild nonmyeloablative condtioning with busulfan

what group is specifically intended to use Kymriah?

patients with Bcell ALL; intended for patients whose cancer has not responded to treatment

what is the third step in retroviral vectors ending in the "producer cell line"?

plasmid with packaging signals introduced into packaging cell line, structural proteins assemble therapeutic gene into virus particles

where is CD19 found?

surface of differentiated Bcells, NOT hematopoietic stem cells or other essentials

what is a liposome vector?

synthetic lipid that binds to and encapsulates plasmid DNA; fuse with cell membrane and deliver genetic payload

the _____________ sequence precedes the hairpin; it confers sequence _______________

target sequence sequence specificity

what makes up the Guide RNA?

target sequence + hairpin structure

t/f gene therapy is highly experimental collection of technologies; lots of potential

true

what is a therapeutic strategy?

vector carries a gene that encodes a protein that is defective/not present in host genes

what is a cytolytic strategy?

vector is designed to kill or eliminate diseased cell/tissue

why is "insertional oncogenesis" considered an unlikely but possible risk for this gene therapy?

vectors cannot reproduce themselves and so cannot repeatedly insert into cell's chromosomes...

when avoiding host immune response, what kind of vectors should be used?

vectors that give minimal/no adverse immune response

what allows for long-term expression of therapeutic drug during cell division?

viral dsDNA insertion into genome

how do they generate a gene therapy vector with retroviruses?

viral genes encoding structural proteins are deleted and replaced with therapeutic gene of interest

what is the fourth step in making retroviral vectors?

virus released from packaging cell to infect new cells; only delivers therapeutic gene into cells