User talk:Hannah Kempton

Template:SiRNA Delivery Across the Blood Brain Barrier to treat Huntington's Disease General overview sentence or two

Background

Blood Brain Barrier

1. The blood brain barrier (BBB) plays a crucial role in modulating cerebral homeostasis and directing neuronal functions. It separates circulating blood from cerebrospinal brain fluid and prevents harmful toxins from reaching the brain.
2. In other parts of the body, the thin endothelial cells lining vessel and capillary walls overlap at leaky junctions that are flexible enough to allow larger molecules like hormones, viruses, and bacteria to squeeze through the junctions or diffuse directly through the cells into the surrounding tissue.
3. However, the endothelial cells lining blood vessels in the brain overlap at tight junctions and are surrounded by a thick basal membrane containing contractile pericytes as well as astrocytic glial cells that provide nutrients and play a role in brain and spinal cord repair. These cells are collectively known as the BBB.
4. The BBB only allows small, essential hydrophobic molecules like O2 and glucose and essential ions like Na+, K+, and Cl- to pass from the blood to the brain and central nervous system (CNS), but prevents the passage of molecules greater than about 500 Da.

Nanodelivery

1. Recently, the use of nanoparticles (NPs) has become increasingly common in solving environmental and medical problems, particularly in targeted drug delivery systems, among others
2. Due to their size, NPs are unique in that their chemical, physical, and biological properties differ from that of their bulk materials, so they can be considered “new” material
3. Significance of Nanoscience in Medicine
   1. Higher surface area:volume ratio → nanomaterials have higher reactivity, mechanical strength, and magnetic or electrical properties
   2. NPs able to cross BBB and mediate repair of BBB damage that may be responsible for diseases like Alzheimer’s
   3. For our purposes, NPs may serve as an effective vector, containing siRNA or other drugs, for targeted drug delivery across the BBB

Significance

The primary purpose of T7.2 is to construct a biological organism that is easier to model than the original biological isolate. There are existing aspects of the natural isolate that make it particularly well suited to system-level analysis. We want to make every effort to preserve these aspects, which include:

1. Viability -- Here viability refers to the ability to propagate the new species for practical experimental purposes.
2. Existing knowledge of the functional genetic elements -- We must continue to harness the tremendous amount of knowledge gained from genetic and biochemical experiments of T7 over the past 60 years. To some extent, these studies are what allow our current models to as detailed as they are.
3. Relatively decoupled from host physiology -- T7 begins shutting off host transcription and solublizing the host genome within minutes of infection. There are very few host proteins necessary for T7 infection. We do not want to purposely or incidentally increase the dependency of the phage upon the host so that we can minimize modeling the intricacies of host physiology.
4. Coupling of entry and transcription -- This coupling leads to the natural organization of genes on the T7 genome. In addition, the timing of genome entry, to some extent, makes modeling the phage easier. In addition, any importance of the ordering of elements on the natural isolate will be more relevant to T7.2.

However, we feel the original biological isolate is not necessarily where we should begin to understand how system-behavior is produced. Specifically, in designing T7.2, the following five goals will drive our design; the first three goals revisit or extend goals motivating the design of T7.1.

1. Our design of T7.2 will enable the unique and selection-independent manipulation of each genetic element via restriction enzymes.
2. We will specify a genome that does not include any functions that might be encoded via the physical coupling of multiple genetic elements.
3. We will specify a genome that only includes elements that we believe contribute to phage gene expression. Moving beyond our design of T7.1, we will actively erase or delete elements of unknown function. In addition, efforts will be made to made to remove unknown genetic elements.
   1. reduced gene sets?
   2. codon shuffling?
4. To attempt to make our modeling of gene expression easier, we will use standard synthetic elements in place of the natural elements that regulate transcription and translation.
5. We will make a genome that is more anemenable to measurements that are important to us, such as adding reporters of transcription and translation.

Taken together, our design of T7.2 should specify a genome that is simpler to model and manipulate, in which we have a putative function for each base pair of DNA involved in phage gene expression. We hypothesize that as a result of the more parsimonious genome design, T7.2 will also encode a dynamic system that is easier to model and interact with, relative to the natural biological isolate.

Meta Considerations

Standardization

We want to standardize certain functional genetic elements to make them easier to model. For example, instead of further characterizing every different ribosome binding site and promoter, we can standardize on a set of that would take considerably less effort to characterize.

Measurement

We want to increase the ability to measure different aspects of phage biology. This may including mRNA and protein reporters, and/or optimizing DpnI restriction sites to ease entry assays.

Manipulability

We need to increase our ability to make selection-independent changes to the genome easily. We will take the some of the lessons we learned in T7.1 about the types and distribution of restriction sites needed.

Encoding

We want to reduce, to as large of an extent as possible, the number of genetic elements that are included in T7.2 that do not encode functions that we do not know about.

Designs

See the Designs Page for further information.