Macrophage therapy

Project Overview

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

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.

• include comparison of "gold standard" for gene therapy techniques?
• find other sources citing macrophages as delivery vectors?

• 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).

Research Problems and Goals

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

Project Details and Methods

Methods used by the Haney Paper:

• • standard cloning with luciferase and gfp reporters
  • transfection
  • mouse macrophage cells, Cath.A neurons
  • Balb mice