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
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
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
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
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
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
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
Critical Plasmids for Safe
Retroviral Production
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 MDRpump 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
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
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
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 orRCR
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
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 outcompetetransduced 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
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
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 bloodCD34+ cells
z5 cytokines, Retronectin
• Plate for MDR PCR +colonies in MC • Inject cells into NOD-SCID
• Analyze NOD-SCID 5-6 weeks later
MDR-Transduced HSC in
NOD-SCID Mouse - MDR PCR
zMethylcellulose colonies: PCR+
zPre- NOD-SCID: 20/30 (66%)
zPost-NOD-SCID:
•Mock: 0/50 + (0%) •A12M1: 16/168 + (10%)Summary: MDR-Transduced
HSC in NOD-SCID Mouse
z MDR transduction of human HSC achievedz 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
VSV-G Envelope Packaging Lines
z High-titerz 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
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
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
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
Sickle Mouse Models
Leboulch Globin Lentiviral
Vector
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