Macrophage therapy

Project Overview

This page is for a 20.109 project by Katie and Emily.

Our initial idea is to use macrophage therapy to target Tay Sach's disease. We would transfect macrophage's with CRIPSR machinery, designed to make 2 double stranded breaks around the mutated hexosaminidase A gene and have a donor dsDNA with the correct version of the gene homologously recombine with the genome. Macrophages are good at crossing the blood brain barrier and are known to transport their genetic material in exosomes efficiently to target cells. Macrophages are also naturally produced by humans, limiting immune response associated with other gene delivery methods such as viruses. Tay Sach's disease already has a substantial animal model since the genomic locus at which the mutation occurred is known. CRISPRs are also the latest genome editing technology, but have yet to come near clinical trials.

Background Information

• Pathology of Neurodegenerative Disorders such as Alzheimers and Parkinsons Disease
  • activation and secretion of reactive oxygen and nitrogen species, leads to oxidative stress, affects function of neurons, astrocytes, and microglia
    • induces ion transport, calcium mobilization, apoptotic programs
  • mitochondrial respiratory chain affects oxidative phosphoyrlation and is responsible for the production of Reactive Oxygen Species
  • observe lack of natural antioxidants such as glutathione and superoxide dismutase, and iron in the SN region of the brain

We can approach curing these diseases by:

• • finding a way to deliver redox enzymes to the brain

How Haney et. al approached cell-mediated drug delivery of antioxidants: 1st Approach:

• used macrophages as cell-carriers since they can carry the antioxidant proteins across the blood brain barrier - to specifically target subregions of the brain
• to stop enzyme degradation – added catalase, packaged with block ionomer complex with cationic block copolymer – producing nanosized particles “nanozymes”
• nanozyme-loaded macrophages administered to mice with brain inflammation facilitated nanozyme transport across blood brain barrier
• also saw prolonged and sustained release of catalase
• saw macrophages migrate from blood away into tissue and unload and supply blood plasma with catalase over 7 days
• macrophages sent nanozyme into cells, caused decomposition of reactive oxygen species, reduced inflammation in the brain – all providing powerful protentation in Parkinson’s Diseased mice.
• Mechanism of transport of nanozyme from macrophage to target cell –
  • uses transient fusion of cellular membranes
  • forms macrophage bridging conduits, filopia, lamellipodia?
  • releases exosomes, vesicles that contain nanozyme
    • recent studies showed that exosomes can be used efficiently for cell to cell transfer
    • show in this paper that incorporated catalase in exosomes altered its localization in the cells of the neurovascular system, enabling it to reach different compartments such as ER, cytoplasm, mitochondria – locations where catalase can deactive reactive oxygen species

2nd approach:

• genetically-modified cell-carriers transfected with plasmid DNA encoding the therapeutic protein
• transfected cells provide sustained expression of protein of interest in the brain
• cell-cariers transfected with neurotropic factors, brain-derived neutotropic factors, glial-cell=line derived neurotropic factor etc etc were delivered to the brain via transfected neural stem cells or bone marrow-derived macrophages for treatment of neurodegenerative diseases

Approach of paper:

• used genetically-modified immunocytes: RAW 264.7 macrophages, transfected with plasmid DNA encoding reporter protein (GFP etc) or therapeutic protein (Catalase)
• gene and protein transfer studied in vitro and in vivo
• demonstrated that systematically administered transfected macrophages release exosomes with incorporated DNA, mRNA, transcription factor molecules and encoded protein in them -→ led to sustained catalase expression → neuroprotection
• key is that cell-mediated drug delivery is promising strategy for targeted transport of therapeutic genes and drugs – missing link for translational gene therapy of inflammatory and neurodegenerative disorders.

Key results of the paper:

• Transfected macrophages were still alive by day 4 and releasing catalase ---> catalase release does not cause cell death
• able to target specifically to the site of the inflammation and not peripheral tissues

• Summary from FNT Assignment: The reduction of brain inflammation is important in the treatment of neurodegenerative diseases. The study below used genetically modified macrophages, carrying reporters and therapeutic genes, in order to upregulate catalase, an enzyme that reduces inflammation. They particularly targeted Parkinson’s disease in mice, and noted a three fold reduction in brain inflammation and a significant improvement in motor functions. This suggests that macrophages have potential as vectors for gene and drug delivery for these types of disorders.

Haney, M et al. (2013), Specific transfection of inflamed brain by macrophages: a new therapeutic strategy for neurodegenerative diseases. PNAS, 8(4).

* Other Possible Targets using Macrophage or Exosome-mediated gene delivery: ** Tay Sach's Disease (our current focus):

• • results from insufficient hexosaminidase A - breakdown cell membrane components that accumulate in nerve cells in the brain
  • no known cure or treatment
  • most patients die by age 4
  • caused by genetic mutation in HEXA gene
  • direct enzyme therapy doesn't work - immune reaction, enzyme too big to be transported into cell

* Exosome Targeting Information - What if we just used exosomes and not macrophages? :

• • What if we just used exosomes to deliver payload and not deal with macrophages
  • Exosomes are hard to target
  • suggestions that ligands on surface of exosomes can specifically interact with target cells
  • recent paper showed that it was possible to use exosomes tagged with a glyocprotein to specifically migrate to neurons.
    • used self-derived dendritic cells to produce the exosomes, engineered dendritic cells to express exosomal membrane protein fused to neuron specific RVG peptide, purified exosomes, electroporated them with siRNA, injected them into mouse neurons and saw gene knockdown

Lee, Y et al. (2012), Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy. Human Molecular Genetics, R1-10.

Alvarez-Erviti, L. (2011), Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature Biotechnology,

*Other Ideas - Involve CRISPRs?:

• • Have macrophage produce non-replicating virus capable of specifically targeting site in genome with mutation
  • Have macrophage / exosomes transmit CRISPR system to Tay Sach's cell - break the gencome where the mutation is and carry correct DNA to be inserted, utilizing cell's natural homologous recombination machinery
  • Results from RNA-Guided Human Genome Engineering via Cas9 by Mali et al have shown some efficiency at getting donor DNA to recombine into places where the CRISPR system was used to make double stranded breaks

• Other Reference Source to be used as background:
  • Burke, B et al. (2002), Macrophages in gene therapy: cellular delivery vehicles and in vivo targets. Journal of Leukocyte Biology, 72(3): 417-28.

Research Problems and Goals

• Challenges with targeting neurodegenerative diseases:
  • limited blood brain barrier permeability
  • inherent peripheral and brain drug permeabilities
  • low therapeutic indices
  • low turnover of neurons

• Cell-Based Gene Targeting
  • noninvasive
  • pass unrecognized by immune system
  • but extremely hard to target exosomes

Project Details and Methods

• Methods used in the Haney et al paper
  • standard cloning, used luciferase / gfp reporters under the CMV promoter. used human catalase as therapeutic gene
  • transferred genetic material into macrophages through transfection (specifically lipofection)
  • cell lines used - mouse macrophage cells (RAW 264.7), Cath.A neurons
  • used live Balb mice
  • analyzed GFP fluorescence using FACS
  • catalase activity measured with Amplex Red assay, measured fluorescence on spectrophotometer
  • Used exosome isolation kit on the cells and then added exosomes to neurons - visualized results with confocal microscopy
  • Used western blot to examine proteins inside the exosomes
  • Exosome lysates were rt-PCRed to measure gene expression
  • 6-OHDA or LPS intoxications given to mice to give a PD or Alzheimer's phenotype
  • mice injected with transfected macrophages

• Gold Standard of Observing microglial movement in a live mouse is by using two-photon laser-scanning microscopy. They imaged in the brain through either a cranial window or a thinned scull so as to not disturb CNS tissue
  • http://ac.els-cdn.com/S0896627312011622/1-s2.0-S0896627312011622-main.pdf?_tid=b29e3c96-bb78-11e2-abc3-00000aacb360&acdnat=1368413964_4ce6d316424dd2b5c81bef6be44ca78c

Background on Macrophages

• located all throughout the body in various tissues in various forms
• through phagocytosis, internalize large particles such as debris, apoptotic cells, pathogens into phagosomes
• mononuclear phagocytic system consists of bone-marrow-derived cells (monocytes & macrophages), having different morphologies, responsible for phagocytosis, cytokine secretion and antigen presentation
  • the system is generated from committed haematopoietic stem cells in the bone marrow
• monocytes are the macrophage precursors, released into circulation, they seed tissues in the spleen, and migrate through the endothelium, where they then differentiate into either macrophages or dendritic cells
• different phenotypes in different parts of the body: microglia (brain), osteoclasts (bone), alveolar macrophages( lungs), Kupffer cells (liver)
  • http://www.nature.com/nri/journal/v11/n11/pdf/nri3073.pdf

• microglia form the innate defensive system of the CNS - present in brain / spinal cord
• upon diseased environment, normal form of microglia convert to activated form
• interact with neurons by recognizing synapses

Background on Targeting

• Natural Macrophage Targeting
• macrophages naturally go towards oxygen-deprived areas of the body - athritis, artheroschleric plaques
• monocytes are recruited out of the bloodstream by chemokines released by tumors. once monocytes reach tumors, undergo phenotypic changes and differentiate into macrophages which promote tumor growth
• hypoxia up-regulates production of chemokines in cells
  • "Importantly, elevated levels of the many of these chemoattractants found in tumors have also been seen in other disease tissues like atherosclerotic plaques and rheumatoid arthritic joints, in which macrophage accumulation in hypoxic areas has been observed" - Murdoch et al. 2004. Mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues. Journal of the American Society of Hematology. 104(8): 2224-2234
• The paper on macrophage targeting Alzheimer's and Huntington's took advantage of a macrophages natural tendency to go toward areas of hypoxia and ability to cross the blood brain barrier. They did not specifically engineer the macrophages to target the neurons.

• How normal microglia migrate:
  • mediated through a purinergic signaling cascade.
  • in vivo experiments by Davalos et al. (2005) showed that 'lowering extracellular ATP concentration by the ATP-hydrolyzing enzyme apyrase results in reduced process movements, whereas artificially created ATP gradients stimulate their motility' Kettemann et al, (2012), Microglia: new Roles for the Synaptic Stripper, Neuron. 77: 10-18.

• Engineering Macrophage Targeting
  • given background information on microglia, perhaps we do not need to engineer the macrophages. The main question now becomes: How to harvest microglia from an organism? Do we know how to differentiate monocytes into microglia?
  • As a backup option - we can explore expressing various glycoproteins on the microglia's exosomes that can help them target neurons specifically, if we see accumulation in other areas of the body
    • Info on exosomes and glycoproteins

• Information on Exosomes and Degradation

• We can extract microglia from a sacrificed mice to use on other mice --> no immune response?

Splitting up the work

Katie:

• project details + methods (4-7 slides of methods & outcomes)
• predicted outcome if stuff goes well / if stuff doesn't go according to plan (1 slide alternative)
• needed resources to complete the work (1 slide)