stem cell applications and good manufacturing practice (gmp) · hiv treatment using gene-modified...

Stem Cell Applications and Good Manufacturing Practice

(GMP)

at the UC Davis Institute for Regenerative Cures

Liver repair and regeneration, bioengineered livers Peripheral artery disease: revascularization to prevent amputation Eye degeneration/blindness Lung disease, lung repair and regeneration Skin: Non-healing ulcers, burn repair Bone repair, osteoporosis, cartilage regeneration Heart disease, infarction repair and stroke Neurodegenerative (Parkinsons, Huntingtons, Alzheimers, ALS) Neurodevelopmental disorders (Autism spectrum, FX-TAS, others) Kidney repair and regeneration Bioengineered bladders, tracheas, and other tissues and organs Blood disorders, autoimmune disorders (Scleroderma, MS) HIV treatment using gene-modified stem cells Hearing, inner ear cilia repair Tumor stem Cells, Cell-based immunotherapy for Cancer

UC Davis Stem Cell Program Disease Teams- 147 Basic, Translational, and Clinical Investigators working together

How are stem cells defined?

Mature Tissues

Differentiation and Commitment

1) Self-renewal 2) Multi-potential 3) Highly proliferative

How are stem cells defined?

TYPES OF HUMAN STEM CELLS -------------------------------------------------------- ADULT TYPE, MULTIPOTENT STEM CELLS, for instance: • Hematopoietic Stem Cells - found in: • Bone Marrow • Umbilical Cord Blood • Mobilized Peripheral Blood Can only make the tissue they are designated to make. There are also many other types of adult type stem cells. -------------------------------------------------------- PLURIPOTENT STEM CELLS • Embryonic Stem Cells (or induced Pluripotent Stem Cells) Can make ALL tissues of the body, but not a complete organism. -------------------------------------------------------- TOTIPOTENT STEM CELLS • Fertilized Oocytes Can make a complete organism.

Hematopoietic stem cell Hematopoietic stem cell

Lineage committed progenitor cell

ADULT TYPE STEM CELLS Scanning electron microscope photograph


HSC

Question we are most commonly asked:

• When will clinical trials using stem cells actually begin?

Answer:

• They BEGAN in 1956.

• First successful bone marrow transplantation from one human to another.

• Stem cells from the donor bone marrow were transplanted to regenerate the blood-forming system of a patient with leukemia.

MALE DONOR

FEMALE RECIPIENT

healthy donor

patient with Leukemia

allogeneic transplant

LEUKEMIA TREATMENT USING ALLOGENEIC BONE MARROW STEM CELL

TRANSPLANTATION

BONE MARROW TRANSPLANT

recipient engrafts with donor cells -develops donor immune system

MALE DONOR

FEMALE RECIPIENT

healthy donor


allogeneic transplant Anti-Cancer Effect 80% CURE RATE of Leukemia


TRANSPLANTATION



MALE DONOR

FEMALE RECIPIENT

healthy donor


allogeneic transplant Anti-Cancer Effect 80% CURE RATE of Leukemia

SIDE EFFECT: anti-recipient reaction (GvHD)


TRANSPLANTATION



Hematopoietic stem cell therapies (expanded)

• Hematopoietic stem cells can be isolated using a cell

surface marker such as CD34 and CD133 or by function of the enzyme Aldehyde Dehydroginase (ALDH). Hematopoietic stem cells have high ALDH expression.

• They can be infused I.V., in concentrated levels, much like a bone marrow transplant, to treat tissue damage.

• They find their own way from the bloodstream to the damaged tissue, especially to hypoxic areas and areas of inflammation, where they accumulate and initiate tissue repair.

Clinical trials currently ongoing Stem cells to treat blindness: Purified human hematopoietic stem cells.

Title: A Pilot Clinical Trial of the Feasibility and Safety of Intravitreal Autologous Adult Bone Marrow Stem Cells in Treating Eyes with Vision Loss from Retinopathy

PI: Susanna S. Park, MD, PhD, Protocol # 305805, UC Davis Department of Ophthalmology & Vision Science.

Purpose: Investigate the feasibility and safety of intravitreal autologous bone marrow stem cell therapy in treating people with irreversible vision loss from retinal degenerative conditions or retinal vascular disorders.

Fifteen subjects with vision loss will be injected intravitreally with autologous CD34 positive cells.

Indication: Patients 18 years of age or older with 20/100 to Count Fingers visual acuity; vision loss due to “dry” age-related macular degeneration, retinitis pigmentosa, retinal vein occlusion, diabetic retinopathy, and hereditary maculopathy.

Hypothesis:

Bone marrow stem cells have regenerative capacity.

They can promote blood vessel growth. They may rescue photoreceptors by

improving blood flow. The patient’s own bone marrow CD34+

cells are safe (no rejection, no tumor formation).

Restoring vision using the patient’s own bone marrow CD34+ cells

In vitro and in vivo experiments were carried out to confirm the hypothesis.

An Investigational New Drug (IND) application was submitted to the US Food and Drug Administration (FDA).

A Phase I clinical study, a SAFETY STUDY with secondary endpoint of EFFICACY was approved by the FDA and initiated by UC Davis.



Patient exam

Outpatient procedure in hospital Bone marrow aspirate

GMP grade magnetic cell isolation

Release tests / QC-QA Transport to treatment area

Cell injection


Start at 8 AM – finish at 5 PM

Patient exam

Outpatient procedure in hospital Bone marrow aspirate

GMP grade magnetic cell isolation

Release tests / QC-QA Transport to treatment area

Cell injection

Study Outcome

A: Fundus photograph prior to treatment B: Fundus photograph 3 months after treatment C: Fluorescent angiogram prior to treatment D: Fluorescent angiogram 3 months after treatment

Study Outcome

SUBJECT 1


Study Outcome

SUBJECT 1

Retinal hemorrhage


Study Outcome

SUBJECT 1

Microaneurysms

Retinal ischemia

Intravitreal Autologous Bone Marrow CD34+ Cell Therapy for Ischemic and Degenerative Retinal Disorders: Preliminary Phase 1 Clinical Trial Findings Susanna S. Park, Gerhard Bauer, Mehrdad Abedi, Suzanne Pontow, Athanasios Panorgias, Ravi Jonnal, Robert J. Zawadzki, John S. Werner, and Jan Nolta IOVS January 2015 56:81

Mesenchymal stem cells have the potential to form bone, cartilage, tendon, fibroblast, fat, and muscle, and may have other very exciting potentials such as contributing to the repair of damaged heart and skeletal muscle, liver, pancreas, kidney, spinal cord, and even brain. Dr. Jan Nolta, since the late 1980s, has pioneered MSC research, and has characterized the in vitro and in vivo characteristics of MSCs. MSCs home into multiple tissues in immune deficient mice, including brain (Meyerrose at al, Stem Cells 2007). Therefore, we may be able to deliver them intravenously to contribute to tissue repair. The possibility of repairing tissues from easily harvested and unwanted fat cells holds broad appeal, and is an intriguing possibility that could have dramatic effect on health care.

Mesenchymal Stem Cells

• In models of acute local injury, MSC preferentially home to, or accumulate in, the damaged tissue (Wu, Nolta et al, Transplantation 2003).

Dr. Nolta’s lab has been

studying these processes for

human stem cells in immune

deficient mouse models and at the molecular level.

Ischemic Limb - 12 hours Ischemic Limb - 48 hours Non-ischemic Limb - 12 hours

Iron nanoparticle-loaded human stem cells are rapidly recruited to a site of ischemic injury

Tail vein injection of 5x105 human marrow stem cells at T = 0 hours

Capoccia et al., 2009

Clinical Trials in Development • Stem cells in peripheral vascular disease: Save limbs from amputation –

planned clinical trial with John Laird using mesenchymal stem cells

Damaged limb- no blood flow Blood flow restored by Stem Cell Infusion

Working toward a clinical trial for CLI following dialog with FDA, IND submission and approval

• MSC from healthy donors will be gene modified to secrete VEGF, and will be injected into limbs of patients with severe ischemia.

• Goal: Prevention of amputation.

• Evaluation of revascularization: Imaging of blood flow and new vessel growth in the affected limb.

• FDA Pre-pre IND meeting successful, pre IND package assembled and to be submitted.

• In vivo studies ongoing to assure safetey and efficacy.

• Clinical trial IND to be submitted 2015, with clinical trial starting after IND approval.

Completed Stem Cell Clinical Trial at UC Davis

Jeffrey Southard, MD I.V. Infusion of ALLOGENEIC

MSCs to repair damage from heart attack (placebo controlled).

Besides tissue repair effects, MSCs have potent immuno-modulatory effects: MSCs do not express MHC Class II molecules (no HLA antigens), and are sheltered from an immune response. Therefore, cellular therapies with allogeneic MSCs are possible.

Trial has been completed at UC Davis.

MSCs (=Prochymal) provided by Osiris.

5 Patients treated.

The role of MSC in the treatment of Huntington’s disease: Translation from pre-clinical to clinical applications

Developed by Dr. Jan A. Nolta Professor, Director of the Stem Cell Program and Institute for Regenerative Cures, University of California, Davis

Neuron generated in vitro Photo courtesy of Paul Knoepfler, UC Davis Stem Cell Program / Shriners Hospital

Huntington's Disease (HD) is a fatal, dominant neurogenetic disorder resulting from a variable length polyglutamine (polyQ) repeat CAG expansion in exon 1of the HD gene.

Repeats confer a toxic gain of function on the protein huntingtin (htt).

Currently, no preventative or curative treatments exist for HD.

Half of the children born to a parent with HD will be affected. Genetic testing of young people living at risk is very controversial, since there is currently no cure.

In addition to new drugs being developed to treat different aspects of the disease such as chorea, cellular therapy and gene therapy provide the best options for permanent cures.

Bone Marrow is harvested From a normal, healthy donor

Cells expanded in a clean room facility

Mesenchymal Stem Cells (MSC): Reparative adult stem cells that can act as excellent

“delivery vehicles” in the body

1987-2010 Nolta lab: Development of models to study biosafety and efficacy of engineered human Marrow Stromal Cells / Mesenchymal Stem Cells (MSC)

Decade-long biosafety studies: Bauer et al., Nolta lab

Mol Ther 16, 1308 (2008)

Studies Underway, Working Toward Cellular Therapy

Trials for Huntington’s disease

1. MSC to test neurorestorative effects. 2. MSC to continually secrete Brain Derived

Neurotrophic Factor (BDNF) to slow down degeneration of neurons

WHY plan to use Mesenchymal stem cells??

• Strong safety profile • Neurorestorative effects

• Relative ease of isolation and expansion

Neurorestorative effects of MSC • Reduce inflammation • Increase vascularization • Reduce death of damaged neurons • Restore synaptic connections between damaged

neurons

MSC can be safely delivered into the brain and spinal cord, in small and large animal models

(Parr et al, 2007, Pittenger 2008, and

Joyce et al, 2010)

• Following intravenous injection, only low levels of

human MSC cross the blood brain barrier in chronic disease models (Meyerrose et al 2007).

• To effectively combat most neurodegenerative diseases we will need to deliver larger numbers of MSC directly into the brain tissue.

• We are validating the biosafety of catheter-based MSC delivery systems into the brains of rodents and non-human primates.

Brain: the final frontier

MSC use the external surface of the vasculature as “train tracks” to migrate throughout the brain tissue to deliver factors to damaged neurons

Medium Spiny Neuron

Damaged/Lost in HD- they control movement, cognition and emotion Kelly, Dunnet and Rosser, Biochem. Soc. Trans. (2009) 37, 323–328

Damaged neurons can “round up” and retract axons: this prevents effective signaling from cell to cell in the neural network

Mesenchymal stem cells can restore synaptic connections between neurons by secreting factors (reviewed in Regenerative Medicine, Joyce et al, Nolta lab, 2010)

MSC migrate to damaged striatum after implantation into the brain in an HD rat model

MSC (dark spots, white arrows) migrated from injection site at the red arrow to the area of striatal damage (white lesion)

Sadan et al, 2009

• Our ongoing studies show that human MSC, injected directly into the brains of immune deficient mice, survive for months and migrate readily throughout the neural tissue.

• MSC are still present in robust numbers and the brain tissue architecture is unaltered.

MSC expanded under GMP conditions were transplanted into the brains of 3 fetal primates under ultrasound guidance

We performed biosafety testing of Intracranial MSC implantation in Non-human primates

Brains and other organs were collected at term- 5 months later

Intra-ventricular MSC transfer in fetal non-human primates. Sonogram in left panel shows route of transfer (arrow) through the coronal suture. MSC were injected at 70 days gestation; (early second trimester) during maximal neuronal proliferation and prior to development of the immune system. (Tarantal Lab, California National Primate Center, UC Davis)

Intracranial injection of human mesenchymal stem cells into fetal non-human primate brain- the most stringent biosafety model available:

1. After 5 months, human mesenchymal stem cells

were still present in the brain tissue.

2. No tumors or other tissue abnormalities were detected.

3. Continued studies of MSC therapy for HD were warranted.

4. CIRM Disease Team grant application followed.

Working toward a clinical trial for HD following dialog with FDA, IND submission and approval

• MSC from healthy donors will be gene modified to secrete BDNF, and will implanted near the affected portion of the brain in symptomatic HD patients.

• Evaluation of neuroprotective effects: slowing of disease progression as measured by Total Functional Capacity score and delay in volumetric MRI changes known to occur in HD. Clinical improvement in severity of movement disorders and cognitive impairment as measured by the Unified HD Rating Scale (UHDRS) and a battery of cognitive tests.

• FDA Pre-pre IND meeting successful, pre IND package assembled and to be submitted.

• In vivo studies ongoing to assure safetey and efficacy.

• Clinical trial IND to be submitted 2015, with clinical trial starting after IND approval.

Skin Repair using Mesenchymal Stem Cells (MSCs) and induced Pluripotent Stem Cells (iPSCs)

• MSC for Non-Healing Ulcers: “Biological Band Aids” (Isseroff/Nolta/Fierro with German partners-

Technical University of Munich). • MSC for Severe Burns: Palmieri, Greenhalgh - Shriners Hospital.

• iPSC for Epidermolysis Bullosa (EB): Growing NEW, gene corrected, intact skin for children with EB.

CIRM disease team - Bauer with Stanford.

TYPES OF HUMAN STEM CELLS

• Hematopoietic Stem Cells found in • Bone Marrow • Umbilical Cord Blood • Mobilized Peripheral Blood = ADULT TYPE, MULTIPOTENT STEM CELLS can only make the tissue they are designated to make. -------------------------------------------------------- • Embryonic Stem Cells (or induced Pluripotent Stem Cells) = PLURIPOTENT STEM CELLS can make ALL tissues of the body, but not a complete

organism. -------------------------------------------------------- • Fertilized Oocytes = TOTIPOTENT STEM CELLS can make a complete organism.

Induced PLURIPOTENT STEM CELLS

• These stem cells are generated from a skin cell or other mature cell of a patient.

• They resemble almost completely naturally occuring embryonic stem cells.

• Can be generated entirely in the lab. • Do not need any cells from an embryo. • Can be differentiated into all tissues of the body.

• Most importantly: Can generate a patient’s own

tissue (no tissue rejection).

Skin fibroblasts

Transduction with 4 genes using gene therapy vectors

Culture of pluripotent stem cells

Differentiation into mature tissues

GENERATION of induced Pluripotent Stem Cells (iPSCs)

Liver Bioengineering Team- Decellularized liver matrix

Zhou and Wu, Nolta and Zern labs 2010

Developing Cell and Organ Replacement for Individual Patients

Patient-specific human Hepatocytes in culture

Induced pluripotent stem cells

Skin fibroblasts- from the patient

Transplant Into patient

Place on scaffold

HEART MUSCLE CELLS DERIVED FROM PLURIPOTENT STEM CELLS

Single cells beating Cell sheet synchronized beating

With pluripotent stem cells, can we make functional neurons? When a part of a rat’s spinal cord gets cut out, the

rat, like a human with a similar injury, will not be able to walk or move the legs.

When neuronal stem cells derived from pluripotent stem cells are inserted into the spinal cord injury site, these stem cells differentiate into functional nerve cells that connect to the ends of the severed spinal cord – and make the paralyzed rats walk again.

Fluorescent double immunolabeling after in vivo grafting reveals that C17.2–NT-3 NSCs (GFP label, green channel) completely fill C3 wire knife lesion cavity (outlined by GFAP immunolabeling, red channel) and migrate for short distances from the graft site in rostral and caudal directions.

Lu P, Jones LL, Snyder EY, Tuszynski MH. Department of Neurosciences, University of California at San Diego, La Jolla 92093-0626, USA. Exp Neurol. 2003 Jun;181(2):115-29.

Pluripotent stem cell derived neurons can repair a massive injury in the spinal cord

Rats with spinal cord defect 5 days after stem cell transplant

Douglas Kerr et al., Johns Hopkins School of Medicine, Journal of Neuroscience, 2003

Rats with spinal cord defect 120 days after stem cell transplant

Douglas Kerr et al., Johns Hopkins School of Medicine, Journal of Neuroscience, 2003

stem cell applications and good manufacturing practice (gmp) · hiv treatment using gene-modified...

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