1. Gene therapy and viral vectors
Lectures for ETE

2. Physical methods of gene delivery
micro-injection
electroporation
gene gun
tattooing
laser
ultrasound

3. DNA Microinjection
Microinjection is the direct-pressure injection of a solution into a cell through a glass capillary.
It is an effective and reproducible method for introducing exogenous material into cells in culture.
Manipulator and Phase-contrast microscope

4. Methodology
Cells: The best cells for microinjecting are large, easily adherent, with a pronounced nucleus, giving them a tall aspect.
In theory, any mammalian cell can be injected although some types provide more challenges than others.
Contractile cells such as muscle often change shape rapidly in response to being injected (particularly when calcium is present in the medium), and cells that do not lay flat when cultured may need to be held in place with a second holding micropipet.

5. Micropipet:
Glass capillary tubing for fabricating micropipet
Micropipette puller for preparing the glass micropipettes
DNA
Any plasmid containing a cytomegalovirus (CMV) promoter-driven reporter gene that may be assayed in individual cells (e.g., green fluorescent protein [GFP] or b- galactosidase) may be used for monitoring the efficiency of microinjection.

6. Microinjection Apparatus:
Microscope
Micromanipulator
Microinjector

7. Applications

8. Animation of microinjection
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9. Electroporation
Electroporation uses controlled, millisecond electrical pulses to create temporary pores in the cell membrane and allow dramatic cellular uptake of a synthetic DNA immunotherapy previously injected into muscle or skin. The cellular machinery then uses the DNA’s instructions to produce one or more proteins associated with the targeted disease. These foreign protein(s), or antigen(s), mimic the presence of an actual pathogen and induce an immune response to provide future protection against the pathogen or eliminate cells infected with an infectious disease or cancer.

12. Applications
Electroporation is the formation of aqueous pores in lipid bilayers by the application of a short (microseconds to milliseconds) high- voltage pulse to overcome the barrier of the cell membrane.
This transient, permeabilized state can be used to load cells with a variety of different molecules including ions, drugs, dyes, tracers, antibodies, oligonucleotides, RNA and DNA.
Electroporation has proven useful both in vitro, in vivo and in patients, where drug delivery to malignant tumors has been performed. In addition, the data show that electroporation of DNA vaccines in vivo is an effective method to increase cellular uptake of DNA and gene expression in tissue leading to marked improvement in immune responses. Electroporation represents a way of increasing the number of DNA-transfected cells and enhancing the magnitude of gene expression, while reducing intersubject variability and requiring less time to reach a maximal immune response compared to conventional intramuscular injection of the vaccine.

13. Gene gun
The gene gun is part of a method called the biolistic (also known as bioballistic) method, and under certain conditions, DNA (or RNA) become “sticky,” adhering to biologically inert particles such as metal atoms (usually tungsten or gold).
By accelerating this DNA-particle complex in a partial vacuum and placing the target tissue within the acceleration path, DNA is effectively introduced.
Uncoated metal particles could also be shot through a solution containing DNA surrounding the cell thus picking up the genetic material and proceeding into the living cell. 
A perforated plate stops the shell cartridge but allows the slivers of metal to pass through and into the living cells on the other side. The cells that take up the desired DNA, identified through the use of a marker gene (in plants the use of GUS is most common), are then cultured to replicate the gene and possibly cloned.
The biolistic method is most useful for inserting genes into plant cells such as pesticide or herbicide resistance.
Different methods have been used to accelerate the particles: these include pneumatic devices; instruments utilizing a mechanical impulse or macroprojectile; centripetal, magnetic or electrostatic forces; spray or vaccination guns; and apparatus based on acceleration by shock wave, such as electric discharge. 

14. Gold particles
Tungsten particles

15. Applications
Another important use of the DNA gun involves the transformation of organelles: chloroplasts, as well as yeast mitochondria. The ability to transform organelles is significant because it enables researchers to engineer organelle-encoded herbicide or pesticide resistances in crop plants and to study photosynthetic processes.
DNA delivery with the gene gun also offers new advantages for research in such areas as DNA vaccination/genetic immunization, gene therapy, tumor biology/wound healing, plant virology and many others.
The major limitations are the shallow penetration of particles, associated cell damage, the inability to deliver the DNA systemically, the tissue to incorporate the DNA must be able to regenerate, and the equipment itself is very expensive.
An objection to this method is that the DNA could be inserted into a working gene in the plant and many of the public worry that this new DNA could then be transferred to wild plants as well and resistance could be conferred to weeds or insects.

18. Lazer Assisted Gene Delivery
The new technique involves the violet laser being focused onto cell membranes for a fraction of a second – this causes the membrane to open up, allowing foreign genes to enter.
The cell's internal mechanism causes the membrane of the cell to heal itself thus appearing to suffer no long-lasting damage.
After inserting the genes, the cells appear to remain healthy and multiply normally.
The presence of the inserted gene in the multiplied cells are then confirmed by observing the red/green fluorescent proteins produced by the ‘new' gene.

20. Lazer induced stress wave (LISW)-based gene-delivery method
Plasmid DNA (circular DNA residing in bacterial cytoplasm) is used as a vector of the gene of interest.
The plasmid is injected into target tissue, on which a laser target is placed, and subject the target to irradiation with a high-intensity, nanosecond laser pulse to induce plasma and hence an LISW.
By placing optically transparent material on the target, the plasma is confined, resulting in an increase in the LISW's impulse.
Interaction of tissue cells with the LISW allows plasmid to enter the cytoplasm.
A black rubber disk is used to which is covered with a polyethyleneterephthalate sheet for the laser target, which is irradiated with a 532nm, 6ns quality-switched neodymium-doped yttrium-aluminum-garnet laser pulse.
Peak pressure of the LISW easily reaches several tens of megapascals, the pulse width is on the order of microseconds, and the pressure is compressive (not tensile), thus enabling minimally invasive tissue interaction.

21.  In vivo targeted gene transfer to various tissues in rodents using LISWs. Green fluorescence indicates expression of an enhanced-green-fluorescent protein gene.

22. Gene delivery through ultrasound waves

23. Nanorods Nanorods are morphology of nanoscale objects.
Each of their dimension ranges from 1–100 nm.
Nanorods may be synthesized from metals or semiconducting materials.
One way for synthesis of nanorods is produced by direct chemical synthesis.
The combinations of ligands act as shape control agents and bond to different facets of the nanorod with different strengths. This allows different faces of the nanorod to grow at different rates, producing an elongated object.

24. Capping agents
Capping agent would help preventing the nanoparticles from growth. Final product could be either a solid or liquid.
Stabilizing agent could be used to prevent agglomeration of the nanoparticles. Here too, the final product could be either a solid or liquid.
Dispersing agent also helps in preventing agglomeration, but the final product must be a liquid.
There may be just one material, for example the PEG, which could serve all the three roles mentioned above. That is the reason why all these terms are being used by the researchers in common.
PEG: Poly Ethylene Glycol

25. Capping/stabilizing agents: ligands, surfactants, polymers, dendrimers, biomolecules
The use of coating nanoparticle with surfactant or polymer is to prevent aggregation of the particles due to nanoparticles high surface energy.
It also controls the size of the particles during synthesis process.
Capping of nanoparticles an be checked using TEM (Transmission Electron Microscope)

26. Addition of biomolecules
After synthesis, the stabilizing agents surrounding the nanoparticles can be replaced by other molecules usually by ligand exchange reactions.
Biomolecules such as DNA/RNA, oligonucleotides (i.e. siRNA, ssDNA), peptides and antibodies, fluorescent dyes, polymers, drugs, tumoral markers, various enzymes and other proteins, that are easily attached to the nanoparticles’s surface.
In addition, ligands can also be linked to the shell of stabilizing agents. One of the most common applications is the linkage of amino groups in biological molecules with carboxyl groups at the free ends of the stabilizing agents.

27. Gold nanorods that detect proteins can be used for kidney disease detection

28. Srikanth Singamaneni, PhD, assistant professor of engineering, along with Evan Kharasch, MD, PhD, and Jerry Morrissey, PhD, at Washington University School of Medicine, have developed a biomedical sensor using gold nanorods designed to detect the elevation of the protein neutrophil gelatinase-associated lipocalin (NGAL), a promising biomarker for acute kidney injury, in urine. Read more: Gold nanorods that detect proteins could simplify kidney disease detection 

30. HIV “Human Immunodeficiency Virus”
A unique type of virus (a retrovirus)
Human body cannot get rid of HIV
Affects specific cells of the immune system, called CD4 cells, or T cells
Get Tested.

31. AIDS Final stage of HIV infection
Occurs when your immune system is badly damaged and you become vulnerable to opportunistic infections.
CD4 cells < 200 cells/mm3 Blood
Without treatment, survival rate is
3 years

32. HIV life cycle

33. Mutations: Intro No standard wild-type strain.
Amino acid differences from one of several wild- type reference sequences.
Reference sequences - laboratory viruses HXB2 and NL43.
These sequences are nearly identical, differing at only a few amino acids not involved in drug resistance.

34. CCR5 European descent Delays the progression of AIDS
In some cases even brings about immunity.
CCR5-delta 32 hampers HIV's ability to infiltrate immune cells.

35. Cont.
The mutation causes the CCR5 co-receptor on the outside of cells to develop smaller than usual and no longer sit outside of the cell.
CR5 co-receptor is like door that allows HIV entrance into the cell.
The CCR5-delta 32 mutation in a sense locks "the door" which prevents HIV from entering into the cell. 

36. D29V Mutation 
Reduced efficacy of highly active antiretroviral therapy (HAART) 
Emerging mutation in HIV-1 protease confer resistance to PIs by inducing structural changes at the ligand interaction site. 

37. Inhibition of HIV

38. Early Inhibitors (fusion inhibitors)
Interfere with binding, fusion and entry of HIV to the host cell by blocking one of several targets.
Maraviroc and Enfuvirtide are the two currently available agents in this class

39. Nucleoside reverse transcriptase inhibitors (NRTI)
Inhibit reverse transcription
RNA is converted into DNA before being integrated into the DNA in the mammalian cell. But these inhibitors don’t allow this transformation.
Zidovudine, Abacavir, Lamivudine, Emtricitabine and Tenofovir.

40. Non-Nucleoside reverse transcriptase inhibitors (NNRTI)
Inhibit reverse transcriptase by binding to an allosteric site of the enzyme.
NNRTIs act as non-competitive inhibitors of reverse transcriptase.
NNRTIs affect the handling of substrate (nucleotides) by reverse transcriptase by binding near the active site.
Nevirapine, Efavirenz, Etravirine and Rilpivirine.

41. Integrase Inhibitor
These medications inhibit integrase, an enzyme that facilitates the insertion of viral DNA into the DNA of infected cells
Example: Raltegravir

42. Protease Inhibitors
Block the viral protease enzyme necessary to produce mature virions upon budding from the host membrane.
Particularly, these drugs prevent the cleavage of gag and gag/pol precursor proteins.

43. Protease Inhibitors (Cont.)
Virus particles produced in the presence of protease inhibitors are defective and mostly non-infectious.
Examples of HIV protease inhibitors are Lopinavir, Indinavir, Nelfinavir, Amprenavir and Ritonavir.