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Disclosure. Gene Therapy. Transfer of genes into cells Expression of transferred genes

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Disclosure

z Equity interest in Genetix Pharm. Inc. z Exclusive license of retroviral cell

lines from Columbia

z No direct participation in MDR clinical

trials

z Columbia U. annual reporting z FDA

Gene Therapy

z

Transfer of genes into cells

z

Expression of transferred genes

zTo correct a defect zTo provide a new function

Gene Replacement/Homologous

Recombination

z

Best theoretical approach

z

Very low efficiency

z

Useful in ES cells

(2)

Gene Addition

z

Best practical approach

z

High efficiency possible

z

Used most often

Vectors for Gene Transfer

z

Naked DNA

z

DNA in lipid complexes

z

Adenoviruses

z

Adeno-associated viruses (AAV)

z

Retroviruses

(3)

Adenoviruses

z Very high titers z Can be used in vivo z Do not integrate; episomal z Are immunogenic and provoke

inflammatory responses

Adeno-associated Viruses

z Hiigh titers

z Can be used in vivo z Variable integration z Are immunogenic

Retroviruses

z Advantages: Acceptable titers and gene

expression; chromosomal integration; stable producer lines available; safety known

z Disadvantages: Require cell division for

(4)

Uses of Gene Therapy

z

Correct genetic defects-ADA,

hemophilia, sickle cell, Gaucher’s

disease

z

Add new gene

functions-angiogenesis, cancer

Gene Therapy Versus Protein

Therapy

z

Potentially permanent correction

with gene as opposed to daily

requirement for drug

z

Must be effective in level of

expression and expression must

be regulatable

Systems to Study Gene

Transfer

z

Tissue culture cells: relatively

(5)

Factor 8 and 9 Deficiencies

z Hemophilia A and B

z Factor 8 and 9 concentrates and

recombinant proteins effective

z Factor 8 and 9 genes in AAV or adenovirus

injected into muscle raises levels in mice and dogs

z Human Factor 9 AAV trial into muscle

underway (High)

z Evidence for immune responses

Ischemic Vascular Disease

z Angioplasty, bypass surgery

available

z VEGFs can grow new blood vessels z VEGF gene as naked DNA injected

into ischemic legs relieves ischemia

z VEGF gene in AAV and adenovirus

injected into ischemic cardiac muscle being tested

Anti-Cancer Gene Therapy

z Add a toxic gene to tumor cells (HSVTK) z Add normal tumor suppressor gene-p53 or Rb z Add anti-sense oligonucleotide to oncogenes

(bcr-abl)

z Provoke immune response to tumor using CD34+ or dendritic cells transduced with antigens

(6)

Adding a Toxic Gene

z Herpes simplex thymidine kinase

(HSVTK)gene:

zSpecifically phosphorylates gancyclovir

and converts it to a toxic product

zEnd result is tumor cell killing zInjected into brain tumors

post-operatively

zPatients treated with gancyclovir zResults equivocal

Anti-Sense to Oncogenes

z

Oligonucleotides with anti-sense

to:

zBCR-Abl in CML zMutated Ras zBCL

zResults to date equivocal

Tumor Suppressor Genes

z

P53

(7)

Increase Anti-tumor Immune

Responses

z

Injecting cytokine genes into

tumors and using as vaccines

z

Adding tumor antigens to

antigen presenting cells

(dendritic cells) and using as

vaccines

Cancer Gene Therapy

z

Protecting marrow cells from the

toxic effects of chemotherapy

z

Use of the multiple drug

(8)

Critical Plasmids for Safe

Retroviral Production

(9)

MDR Gene Therapy

z MDR gene product is a p-glycoprotein z Pumps natural compounds out of cells z Many classes of anti-cancer drugs require MDR

pump for removal

z Normal marrow cells have little or no MDR gene function

z Add a normal MDR gene to marrow stem cells z Provides drug resistance

(10)
(11)

MDR Transduction in Mice

z

MDR gene present and expressed

up to one year

z

Evidence for stem cell

transduction

z

Taxol selects MDR-transduced

cells

Challenges of Human Gene

Therapy

z

Complete safety

z

Unique receptors on human HSC

z

High level and efficient gene

(12)

Autotransplantation

z

Harvest stem cells from patient

z

Transduce stem cells with vector

containing gene of interest

z

Return transduced stem cells to

patient

Peripheral Blood Stem Cells

z Capable of marrow reconstitution z Easily harvested by out-patient apheresis z Mobilized with chemotherapy/growth

factors

z Efficiently transduced z Repeated harvesting and use

z Cells of choice for marrow transplantation

Progenitor Assays

z

Methylcellulose plates

(13)

Transduction Protocol

z CD34+ cells cultured on fibronectin

plates with IL-3, IL-6 and SCF

z 48 hr pre-incubation z Two changes of retroviral

supernatant over 24 hrs

z Successful MDR transduction of

methylcellulose colonies

zResistance to taxol

Summary

z These results indicated the feasibility

of using CD34+ PBPC MDR transduction to provide drug resistanceof marrow in Phase 1 clinical trials

Columbia MDR Phase1Clinical Trial

z Safety demonstrated: no delayed engraftment or

RCR

z Feasibility shown: Large scale retroviral supernatants and CD34+ cells used in scale-up z Pre-infusion: High-level CD34+ transduction in

BFU-E and CFU-GM

(14)

Requirements for HSC Gene

Transfer

z Stem cells required for short- and

long-term marrow repopulation

z Progenitors (BFU-E and CFU-GM) are

irrelevant to repopulation

z True stem cells (NOD-SCID) required

for marrow homing, marrow repopulation and expansion

Murine Studies-Qin 1999

z Untransduced (fresh) cells outcompete

transduced cells for marrow engraftment both short- and long-term

z Two to 4 day delay in infusing untransduced cells after infusing transduced cells increases short-and long-term repopulation of transduced cells

Indiana Trial- MDR Gene Therapy

z Pts with relapsed germ cell tumors z Intensive carboplatin and etoposide

(15)

MDR-Indiana Gene Therapy Trial

z Best results reported to date of HSC

gene therapy

z MDR-transduced cells persist up to 1

year and are selectable with drug

z TPO, SCF and G-CSF are best growth

factor combination

(16)

Indiana Trial: Summary

z Best HSC gene transfer and

expression to date

z MDR-transduced cells selected by

chemotherapy

z Retronectin effect positive

z TPO, SCF, G-CSF growth factors best z Lack of competition of fresh and

transduced cells critical

NOD-SCID Mouse Assay

z Only valid assay for human HSC z MDR-transduce human cord blood

CD34+ cells

z5 cytokines, Retronectin

• Plate for MDR PCR +colonies in MC • Inject cells into NOD-SCID

• Analyze NOD-SCID 5-6 weeks later

(17)

MDR-Transduced HSC in

NOD-SCID Mouse - MDR PCR

z

Methylcellulose colonies: PCR+

z

Pre- NOD-SCID: 20/30 (66%)

z

Post-NOD-SCID:

Mock: 0/50 + (0%)A12M1: 16/168 + (10%)

Summary: MDR-Transduced

HSC in NOD-SCID Mouse

z MDR transduction of human HSC achieved

z Transduction efficiency comparable

to that of clinical trial:1-10% of human cells

z Conditions: 5 cytokines, no

polybrene, Retronectin, multiple viral exposures

Amphotropic Retroviral Packaging

Lines

z AM12 et al

z Titers between 104 and106

z Limited receptor expression on human

HSC

z Cannot be concentrated

(18)

VSV-G Envelope Packaging Lines

z High-titer

z Virus can be concentrated

z Transient packaging due to VSV-G toxicity

z Adding plasmids to 293T cells

z Plasmids require SV40 T antigen expression

z Variable packaging and titers z Potential recombinational events

z Difficult to scale-up as compared to stable lines

RD114 Envelope Packaging Lines

z Transient supernatants produced z High-titer

z Can be concentrated

z Efficiently transduce human HSC as

tested in NOD-SCID mice (Kelly et al 2000, Gatlin et al 2001)

Stable RD114 Packaging Line (M.

Ward)

z Moloney gag-pol in 3T3 cells

z Add RD114 gene with phleomycin selection z Isolate high titer clones with NeoR gene and G418

(19)

Current Bank lab GT

Goals-2003

z Better HSC transduction - new envelopes (RD114); transient VSV-G packaging lines

z Concentrate on human globin gene therapy using Leboulch lentiviral vector

z Use NOD-SCID mouse model to predict human HSC transduction

Cure of Children with

X-SCID

z Most successful human trial to date

z T cells lack γC cytokine receptor required for lymphoid

proliferation

z Retroviral transfer of γC cytokine receptor gene into CD34+

cells

z Autotransplantation z Selection of corrected cells

z Normal immune function in 7/9 patients z T cell leukemia (clonal) in 2/9 patients 3 years

post-transduction

Leukemia in Children with

X-SCID

z Similar insertional mutagenesis events in both children

z Unregulated γC cytokine receptor gene inserted into LMO2 locus

z Activation of LMO2, a proliferative gene

(20)

Lentiviral Vectors

z Transduce non-dividing cells

z Can transduce murine and human HSC

efficiently

z Very high titers

z Better for globin gene therapy

z Can cure mouse models of human sickle

and thalassemia

z Safety issues

Lentiviral Vector Plasmids

(21)

Successful

β Thal Gene Therapy

z May et al: Nature 2000 z β globin gene correction in β

thalassemic mice

z Lentiviral vectors with extensive β

-LCR elements used

z Gene-modified cells produce β globin

in vivo

z Correction of thalassemia phenotype

Successful Sickle Gene Therapy

z Pawliuk et al: Science 2001 z β globin gene correction in two

mouse models of sickle cell

z Lentiviral vectors with extensive β

(22)

Sickle Mouse Models

Leboulch Globin Lentiviral

Vector

(23)

Current Gene Therapy

Experiments - 4/03

z Viruses with new envelopes - RD114 z New incubation conditions- BIT media,

new cytokines

z NODSCID mouse assay for true HSC

-CD34+ CD38- cells

z Use of lentiviral vectors in human globin

References

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