Disclosure

	Equity interest in Genetix Pharm. Inc.
	Exclusive license of retroviral cell lines from Columbia
	No direct participation in MDR clinical trials
	Columbia U. annual reporting
	FDA

Gene Therapy

	Transfer of genes into cells
	Expression of transferred genes
		To correct a defect
		To provide a new function

Gene Replacement/Homologous Recombination

	Best theoretical approach
	Very low efficiency
	Useful in ES cells
	Not practical at present

Gene Addition

	Best practical approach
	High efficiency possible
	Used most often

Vectors for Gene Transfer

	Naked DNA
	DNA in lipid complexes
	Adenoviruses
	Adeno-associated viruses (AAV)
	Retroviruses
	Lentiviruses

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Adenoviruses

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

Adeno-associated Viruses

	Hiigh titers
	Can be used in vivo
	Variable integration
	Are immunogenic

Retroviruses

	Advantages: Acceptable titers and gene expression; chromosomal integration; stable producer lines available; safety known
	Disadvantages: Require cell division for stable integration
	Uses: Bone marrow stem cell gene therapy
	Lentiviruses better

Uses of Gene Therapy

	Correct genetic defects-ADA, hemophilia, sickle cell, Gaucher’s disease
	Add new gene functions-angiogenesis, cancer

Gene Therapy Versus Protein Therapy

	Potentially permanent correction with gene as opposed to daily requirement for drug
	Must be effective in level of expression and expression must be regulatable

Systems to Study Gene Transfer

	Tissue culture cells: relatively easy
	Mice
	Larger animals - dogs, primates
	Humans

Factor 8 and 9 Deficiencies

	Hemophilia A and B
	Factor 8 and 9 concentrates and recombinant proteins effective
	Factor 8 and 9 genes in AAV or adenovirus injected into muscle raises levels in mice and dogs
	Human Factor 9 AAV trial into muscle underway (High)
	Evidence for immune responses

Ischemic Vascular Disease

	Angioplasty, bypass surgery available
	VEGFs can grow new blood vessels
	VEGF gene as naked DNA  injected into ischemic legs relieves ischemia
	VEGF gene in AAV and adenovirus injected into ischemic cardiac muscle being tested

Anti-Cancer Gene Therapy

	Add a toxic gene to tumor cells (HSVTK)
	Add normal tumor suppressor gene-p53 or Rb
	Add anti-sense oligonucleotide to oncogenes (bcr-abl)
	Provoke immune response to tumor using CD34+ or dendritic cells transduced with antigens

Adding a Toxic Gene

	Herpes simplex thymidine kinase (HSVTK)gene:
		Specifically phosphorylates gancyclovir and converts it to a toxic product
		End result is tumor cell killing
		Injected into brain tumors post-operatively
		Patients treated with gancyclovir
		Results equivocal

Anti-Sense to Oncogenes

	Oligonucleotides with anti-sense to:
		BCR-Abl in CML
		Mutated Ras
		BCL
		Results to date equivocal

Tumor Suppressor Genes

	P53
	Retinoblastoma (RB)

Increase Anti-tumor Immune Responses

	Injecting cytokine genes into tumors and using as vaccines
	Adding tumor antigens to antigen presenting cells (dendritic cells) and using as vaccines

Cancer Gene Therapy

	Protecting marrow cells from the toxic effects of chemotherapy
	Use of the multiple drug resistance gene

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Critical Plasmids for Safe Retroviral Production

MDR Gene Therapy

	MDR gene product is a p-glycoprotein
	Pumps natural compounds out of cells
	Many classes of anti-cancer drugs require MDR pump for removal
	Normal marrow cells have little or no MDR gene function
	Add a normal MDR gene to marrow stem cells
	Provides drug resistance
	Can also be used to select transduced cells

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MDR Transduction in Mice

	MDR gene present and expressed up to one year
		Evidence for stem cell transduction
	Taxol selects MDR-transduced cells

Challenges of Human Gene Therapy

	Complete safety
	Unique receptors on human HSC
	High level and efficient gene transfer

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Autotransplantation

	Harvest stem cells from patient
	Transduce stem cells with vector containing gene of interest
	Return transduced stem cells to patient

Peripheral Blood Stem Cells

	Capable of marrow reconstitution
	Easily harvested by out-patient apheresis
	Mobilized with chemotherapy/growth factors
	Efficiently transduced
	Repeated harvesting and use
	Cells of choice for marrow transplantation

Progenitor Assays

	Methylcellulose plates
	Measure BFU-E and CFU-GM
	PCR-positive colonies
	Colonies with and without taxol

Transduction Protocol

	CD34+ cells cultured on fibronectin plates with IL-3, IL-6 and SCF
	48 hr pre-incubation
	Two changes of retroviral supernatant over 24 hrs
	Successful MDR transduction of methylcellulose colonies
		Resistance to taxol

Summary

	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

	Safety demonstrated: no delayed engraftment or RCR
	Feasibility shown: Large scale retroviral supernatants and CD34+ cells used in scale-up
	Pre-infusion: High-level CD34+ transduction in BFU-E and CFU-GM
	Post-infusion: 2/5 patients with low level MDR PCR + cells

Requirements for HSC Gene Transfer

	Stem cells required for short- and long-term marrow repopulation
	Progenitors (BFU-E and CFU-GM) are irrelevant to repopulation
	True stem cells (NOD-SCID) required for marrow homing, marrow repopulation and expansion

Murine Studies-Qin 1999

	Untransduced (fresh) cells outcompete transduced cells for marrow engraftment both short- and long-term
	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

	Pts with relapsed germ cell tumors
	Intensive carboplatin and etoposide therapy followed by either MDR-transduced or untransduced HSC
	Three cycles of oral etopside
	CH-296 fibronectin fragment (Retronectin)
	Abonour-Nature Medicine 2000

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Indiana Gene Therapy Trial

	Best results reported to date of HSC gene therapy
	MDR-transduced cells persist up to 1 year and are selectable with drug
	TPO, SCF and G-CSF are best growth factor combination
	Retronectin fragment used

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Indiana Trial: Summary

	Best HSC gene transfer and expression to date
	MDR-transduced cells selected by chemotherapy
	Retronectin effect positive
	TPO, SCF, G-CSF growth factors best
	Lack of competition of fresh and transduced cells critical

NOD-SCID Mouse Assay

	Only valid assay for human HSC
	MDR-transduce human cord blood CD34+ cells
		5 cytokines, Retronectin
	Plate for MDR PCR +colonies in MC
	Inject cells into NOD-SCID
	Analyze NOD-SCID 5-6 weeks later

NOD-SCID Mouse Engraftment

MDR-Transduced HSC in
NOD-SCID Mouse - MDR PCR

	Methylcellulose colonies: PCR+
		Pre- NOD-SCID: 20/30 (66%)
		Post-NOD-SCID:
			Mock: 0/50 + (0%)
			A12M1: 16/168 + (10%)

Summary: MDR-Transduced HSC in NOD-SCID Mouse

	MDR transduction of human HSC achieved
	Transduction efficiency comparable to that of clinical trial:1-10% of human cells
	Conditions: 5 cytokines, no polybrene, Retronectin, multiple viral exposures

Amphotropic Retroviral Packaging Lines

	AM12 et al
	Titers between 104 and106
	Limited receptor expression on human HSC
	Cannot be concentrated
	Safety and scale-up documented in human clinical trials
	Low-level transduction efficiency in human clinical trials

VSV-G Envelope Packaging Lines

	High-titer
	Virus can be concentrated
	Transient packaging due to VSV-G toxicity
		Adding plasmids to 293T cells
		Plasmids require SV40 T antigen expression
	Variable packaging and titers
	Potential recombinational events
	Difficult to scale-up as compared to stable lines

RD114 Envelope Packaging Lines

	Transient supernatants produced
	High-titer
	Can be concentrated
	Efficiently transduce human HSC as tested in NOD-SCID mice (Kelly et al 2000, Gatlin et al 2001)

Stable RD114 Packaging Line (M. Ward)

	Moloney gag-pol in 3T3 cells
	Add RD114 gene with phleomycin selection
	Isolate high titer clones with NeoR gene and G418
	Make retroviral supernatants
	Concentrate virus by centrifugation
	Can transfer G418 resistance to human CD34+ cells
	Can transfer normal b globin gene into sickle CD34+ cells

Current Bank lab GT Goals-2003

	Better HSC transduction - new envelopes (RD114); transient VSV-G packaging lines
	Concentrate on human globin gene therapy using Leboulch lentiviral vector
	Use NOD-SCID mouse model to predict human HSC transduction

Cure of Children with X-SCID

	Most successful human trial to date
	T cells lack gC cytokine receptor required for lymphoid proliferation
	Retroviral transfer of gC cytokine receptor gene into CD34+ cells
	Autotransplantation
	Selection of corrected cells
	Normal immune function in 7/9 patients
	T cell leukemia (clonal) in 2/9 patients 3 years post-transduction

Leukemia  in  Children with X-SCID

	Similar insertional mutagenesis events in both children
	Unregulated gC cytokine receptor gene inserted into LMO2 locus
	Activation of LMO2, a proliferative gene
	A rare event in an early T cell/HSC that leads to a leukemic transformation
	Slow growth and eventual proliferation of the clone
	May be prevented by regulated gC cytokine receptor gene

Lentiviral Vectors

	Transduce non-dividing cells
	Can transduce murine and human HSC efficiently
	Very high titers
	Better for globin gene therapy
	Can cure mouse models of human sickle and thalassemia
	Safety issues

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Lentiviral Plasmids

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Successful b Thal Gene Therapy

	May et al:  Nature 2000
	b globin gene correction in b thalassemic mice
	Lentiviral vectors with extensive      b -LCR elements used
	Gene-modified cells produce b globin in vivo
	Correction of thalassemia phenotype

Successful Sickle Gene Therapy

	Pawliuk et al:  Science  2001
	b globin gene correction in two mouse models of sickle cell
	Lentiviral vectors with extensive      b -LCR elements used
	Gene-modified cells produce b globin in vivo
	Correction of sickle phenotype

Sickle Mouse Models

Leboulch Globin Lentiviral Vector

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Current Gene Therapy Experiments - 4/03

	Viruses with new envelopes - RD114
	New  incubation conditions- BIT media, new cytokines
	NOD-SCID mouse assay for true HSC - CD34+ CD38- cells
	Use of lentiviral vectors in human globin gene therapy