Exam II

Lentivirus

RNA viral method for gene delivery by ssRNA derived from HIV but replication incompetent

Genome integration & stable expression for proliferating cells

+Long term, stable, largeish (8-9kB), dividing and nondividing cells

-Insertional mutagenesis, complex production

Adenovirus

DNA virus discovered from human adenoid tissue that naturally infects respiratory cells, using dsDNA as a gene delivery vector

No genome integration

+Easy production, high transduction efficiency, all cell types, up to 35kB insertional capacity

-Can trigger strong immune response at high dosage(inflammation, organ toxicity, death), transient gene expression (several weeks, not ideal for long-term correction)

Adeno-Associated Virus (AAV)

ssDNA method that uses a helper virus (adenovirus), or helper plasmids

Sometimes integrates into genome

+Safe, stable long-term (years), nonpathogenic, transduces dividing and nondividing cells efficiently, low immunogenicity compared to adenovirus

-Small insertional capacity (5kB), complex production, low yield & expensive scaling up

Transduction

A viral-mediated gene delivery process where a virus introduces genetic material into host cells

Transfection

The general process of introducing nucleic acid plasmids to mammalian cells, often using nonviral methods, to produce viral particles/proteins

In silico

virtual, computer setting, simulation

In vitro

in a lab dish or test tube outside of the body

In vivo

In a living organism, inside the body

In situ

in the original tissue without removal

Ex vivo

cells are removed, edited, put back

Viral gene delivery methods

Cell reprogramming approach using viral factors delivered as single or double stranded DNA or RNA

-Lentivirus

-Adenovirus

-AAV

Non-viral systems

Gene delivery methods that deliver reprogramming factors without a virus

-Lipid nanoparticles (LNPs)

-Electroporation

Virus production process

Transformation, Transfection, Tranduction

Transformation

The process of introducing plasmid DNA into bacterial cells so that they can replicate and produce large quantities of the plasmid

-used during cloning and amplification steps

Transformation

The process of introducing plasmid DNA into bacterial cells so that they can replicate and produce large quantities of the plasmid

-used during cloning and amplification steps

Lipid Nanoparticle (LNP)

Self-assembled spheres made up of: ionizable lipids, phospholiipids, cholesterol, and a PEG-lipid coating

-different component roles that form a stable sphere that carries our cargo: mRNA or CRISPR

Ionizable lipid LNP

positive charge at low pH that binds to negative RNA and neutral at physiological 7.4 pH that reduces toxicity and won’t disrupt cell membranes or cause immune activation

PEGylated lipid LNP

hydrophilic coating, evades immune detection and body clearance, increases circulation time, allowing more particles to reach target cells

Cholesterol

fills gaps between other lipids and controls flexibility, stabilizes particle, improves mRNA delivery

Phospholipid

stabilizes lipid bilayer and aids in membrane fusion during endosomal escape

LNP Mechanism

1) Cellular uptake (endocytosis): LNP binds to membrane and gets engulfed into endosome

2) Endosomal escape: acidic environment activates ionizable lipids in LNP, disrupts endosome, cargo releases and destabilizes membrane

3)Cargo release: once free, LNP releases mRNA or CRISPR, able to be translated or perform editing

Systemic Delivery

delivery to blood to circulate the bloodstream and reach many tissues and organs

EX: LNPs interact with macrophages, endothelial cells, and enzymes that degrade RNA

-can be delivered through intravenous injection (IV) to blood or orally administered

-common for gene therapy or LNP delivery

Local Delivery

Delivery directly to a specific site, primarily targeting a specific tissue

EX: LNPs stay near injection site and are taken up by dendritic cells or muscle cells

-commonly delivered through intramuscular or intradermal injection

A. Endocytosis→Endosomal escape→Cargo release

B. Because transduction requires a virus to deliver genetic material

Virus needed (uL)= 7uL x (200,000 cells/15,000 cells) = 93uL virus

You need long-term gene expression in non-dividing neurons in the retina.

The gene is small (~3 kb), and safety is critical.

Which viral vector would you choose?

Because safety is critical, AAV’s are the best option, and they have smart insertional capacity on top of stable long-term gene expression in dividing and nondividing cells

You’re testing a new gene that might promote cell differentiation.

You don’t need to inject into animals yet,  just show stable integration in dividing cells. What vector would you use?

Because you need stable gene integration that promotes cell differentiation, lentivirus is the best option

You want strong, short-term expression of a viral antigen to trigger an immune response. What vector would you use?

Adenovirus since it has high transduction efficiency with short-term transient gene expression that only lasts a few weeks and triggers a strong immune response

Electroporation

the use of electricity to open cell membranes temporarily allowing negatively charged DNA to move toward positive cell and enter through pores

done by combining host cells with nucleic acids needed for insertion within conductive solution-electroporation buffer that lets electric current pass evenly through sample without frying cells

electric field disturbs the phospholipid bilayer, temporarily forming pores in cell membrane, then it is allowed to recover

+easy, rapid, stable, transfect large number of cells in short time, all cell types

-high cell death/mortality as a result of high voltage pulse leaving cells unable to recover membrane

C. It uses a short electrical pulse to form temporary pores in the membrane

Engineered Nucleases

2 types:

Zinc Finger Nucleases (ZFNs)

Transcription Activator Like Effector Nucleases (TALENS)

2 parts:

1) Binds the DNA

2) Nuclease- like scissors that cut DNA and make double stranded break

ZFNs

DNA recognition: each zinc finger protein binds to 3 DNA bases-one amino acid-several fingers linked together to target a unique DNA sequence

fokl enzyme as scissors, DNA cleavage domain

zinc finger: DNA binding domain, work in pairs, each binding one site of target DNA, fokl dimerizes and cuts DNA between them, double stranded break created repaired by cell, allowimg gene knockout or insertion

TALENS

DNA recognition: TALE binds 1 specific DNA base for precise targeting of any sequence

fokl scissors cleavage domain: TALENS work in pairs binding opp sides of target DNA, dimerizing and cutting DNA between them

double stranded break created and repaired by cell, allowing gene knockout or gene insertion

CRISPR-Cas9

specificity of gene editing with guide RNA that recognizes DNA by complementary base pairing, allowing programmability and precision

w Cas9, gRNA, PAM

Cas9

scissors for dsDNA

gRNA

GPS address that brings Cas9 to exact DNA sequence needed to be edited, new gRNA design for each new target

PAM

short DNA sequence for Cas9 binding

D. AAV, HDR

HDR

homology directed repair that uses a donor DNA template to insert or correct sequences by gene knockout in

NHEJ

non homologous end joining: a quick fix that introduces small insertions or deletions for a gene knockout, default when no donor

Insulin vs CRISPR

CD47 overexpression to help cells avoid immune detection, don’t eat me signal to block innate immune system

Knockout of MHCI,II to block adaptive immune system attack

C

Biomaterial

all substances other than food and drugs that are utilized for biomedical purposes:

1) to treat or diagnose illnesses

2) to repair, enhance, or replace tissues, organs, or physiological functions

Biomaterial Requirements

Biocompatibility & Bioactivity: how well material interface or integrates w live tissue

Biodegradability & Corrosion: natural removal and rate, toxicity, byproducts, body elimination

Mechanical Properties: integrity throughout use and strength

Microscopic & Macroscopic Architecture: shape and size of bulk structure and porosity, fiber diameter and length

Fabrication & Cost: complexity and expensiveness

Biocompatibility and Bioactivity

cells need to adhere and migrate, proliferate, produce ECM, normal function

pores allow for binding sites on surface as they mimic ECM scaffolds, where adhesion proteins can be absorbed

synthetic scaffolds may require protein absorption

Biodegradability

may need to be degradable or not depending on time needed in body

scaffold degradation rate must match natural regeneration (healing) as closely

allows controlled release of drugs

must exit body safely

Mechanical Properties

strength persistent indefinitely until degradation or replacement

surgically resistant

compliance or brittle character match implant site

porosity and fibrosity for adhesion and integration

Young’s Modulus (E) / Elastic Modulus

slope of curve at elastic region resembling stiffness

stress/strain

Yield stress/strength

point where material will experience plastic deformation or permanent deformation afterwards w more strain

upper: higher stress point separating elastic and plastic regions

lower: lower stress in plastic region before extra stress leads to max strength

Ultimate Strength/ tensile/ max

capacity of material to withstand loads to elongate at peak stress point in plastic region

Rupture strength/fracture point

max stress a material can withstand before breaking at end of line

Toughness

ability of a material to absorb energy and plastically deform without fracturing -area under curve, integrate

Brittle material

strong w little strain for high stress

sudden fracture w little or no plastic deformation

ex: glass

Strong but not ductile

Ex: steel wires

Stretch very little and break suddenly

Ductile material

Past elastic region, strange section where necking happens -a permanent deformation in plastic region

Plastic material

Very small elastic region, no steepness, straight across strain with small stress

Measuring mechanical properties

Brittle, strong but not ductile, ductile, plastic

Extracellular matrix (ECM)

Heterogenous structure w functional proteins like collagen, fibronectin, laminin, plus grown factors, proceoglycams, and polysaccharides

- porous and fibrous

- wide stiffness range depending on tissue type

Why make scaffolds porous and fibrous

Mimic natural ECM

Integrate with cells for growth and regeneration, providing space for cells to maneuver and high surface area for adhesion

Composed of synthetic and natural polymers

Can be used to grow tissue in vitro or as catalyst for regeneration in vivo

Micro and macroscopic architecture

Pore sizes and interconnected structures, distribution, shape, roughness, porosity affect:

migration and penetration

adhesion

proliferation

diffusion of nutrients and waste

uniform distribution

vascularization

Porisometry

Measured by:

SEM (surface spread w electrons cross sectioned)

liquid displacement

CT

Fluid Flow Modeling

Organic Biomaterials

Natural polymers

Synthetic polymers

Inorganic Biomaterials

Metals

Ceramics

Metal Alloys examples

Ex: Stent, a metal mesh or fiber in place for blockages

Ex2: artificial joints

Titanium alloy

Vascular stent, fibrous mesh, combination of two or more elements, at least one being a metal

Properties:

Mech-strong nd stiff

Biocom-little interactions with body

Degra-none

Fabrication-by laser machining

Chromium

Solution to metal corrosion that serves as a protective layer, nanodiamond does the same and both prevent metallosis buildup of toxic metal ion debris in soft tissue resulting from body environment corrosive to metals

Metal Alloy Fabrication Methods

Thermo-mechanical: heating and shaping

Powder Metallurgy: metal powder compacted into porous material EX: titanium scaffold for bone regeneration

Laser machining: metal carved with lasers EX: laser machined nitinol vascular stent of nickel and titanium

Metal Alloys

• + good mechanics (strength, compression, fatigue and wear resistance)

• +corrosion resistant

• +nonreactive in body

• -unnatural Elastic modulus (stiffness-extremely, much greater than bone)-causes breakdown resorption and necrosis at bone-metal interface

• -not biodegradable or slow

• -no well integration with body

-Titanium: pure metal itself

-other options are combinations

-but can be used in alloys

Bioceramics

ceramic material engineered to interface with biological systems:

bioinert

bioactive

bioresorbable

EX: HA-bone component naturally occurring with high bioactivity

TCP-natural, high bioactivity

Bioactive glass-silicate w metals and minerals

Applications: dental and bone fillings, coatings for implant-bone interfaces, musculoskeletal supports

Hydroxyapatite (HA)

Porous bioceramic material crystals can be implanted to bone cavity for regeneration facilitation

Bioceramic Properties

• +bone compatibility because similar elastic moduus, chemically and structurally similar, natural ceramics that enhance osteoblast differentiation and proliferation in vitro

• +corrosion resistant

• +compression resistant

• -brittle, limited or no plastic deformation

• -difficult to shape

Chemically and structurally similar to bone

Corrosion resistant

Compression resistant

Biodegradable Polymers

Natural-completely degradable, 3 natural proteins for natural-based and san, ose,tran polysaccharides

Synthetic-partially degradable-polys and phos

Hydrogels

Hydrophilic polymers that form bonds with surrounding liquid and crosslink with each other

-like ECM and soft tissue mech and structure

-biodegradable

-applications to cell culture, scaffolds, building blocks

Ex: collagen, gelatin, alginate, Poly lactic acid (PLA)

Natural Polymers

• +biocativity due to many proteins of natural ECM, promotes cell adhesion and proliferation

• +can be remodeled by body

• +biodegradable

• -batch vaariability

• -poor mechanics

• -expensive synthesis

Poly-L-Lactic Acid Vascular Stent (Synthetic Polymer)

Mechanics: low stiffness and strength than metal

Biocompatibility: not as much as naturally derived polymers, some interaction

Degradability: hydrolytic

Synthetic Polymers Properties

• +cheaper fabrication, no variability, long shelf life, more than natural

• +wide tunability of mechanics

• +biodegradable

• -less bioactivity than natural polymers

Bioactivity

Composite Materials

combined materials

ex: tunable mechanics of synthetic polymers combined with bioactivity of natural polymers

ex: polymer and ceramic brittleness with elasticity

ex: ti dental implant with bioactive glass mechanics of titanium with ceramics bioactivity

Solvent Casting & Porogen Leaching

creates porous scaffolds

1) Porogens like salt crystals fat or sugar placed in mold with soluble solvent

2) Porogen Leaching: embedded porogens dissolved out with solvent like water or alcohol

-size and architecture of pores controlled by porogen size and shape

-difficult to control consistency and interconnectivity of pores

Electrospinning/Electrospraying

1) Voltage applied to capillary tube ejector

2) Soluble polymer ejected to collector and drawn to fiber or sprayed (viscosity and voltage-dependent)

3) Solvent evaporation with thin fiber of polymer left behind

CONS

-time consuming

-