1. Chapter 6-Nucleic Acids as Therapeutic Agents
Targeting specific mRNA and DNA sequences
Antisense RNA
Aptamers
Ribozymes and DNAzymes
Interfering RNA or RNAi
Zinc finger nucleases
CRISPR-Cas 9 system
Nanozymes
Nanoparticles
Viral delivery systems
Nonviral delivery systems
Direct injection, lipids, bacteria, dendrimers, antibodies, aptamers, transposons
Gene therapy
Prodrug activation therapy and Promoterless gene targeting

2. Figure 6. 1 Schematic representation of A. In vivo gene therapy and B
Figure 6.1 Schematic representation of A. In vivo gene therapy and B. Ex vivo gene therapy

3. Figure 6.2 Inhibition of translation of specific mRNAs by antisense (AS) nucleic acid molecules. A. Antisense cDNA and B. Antisense oligonucleotide

4. Figure 6.5 Aptamers are nucleic acid sequences, either RNA or DNA, that can bind and inhibit proteins, amino acids, drugs, or other molecules.

5. Figure 6.9 Ribozymes are catalytic RNA molecules that cleave target (substrate) RNAs. A. Hammerhead ribozyme- mRNA substrate complex and B. Hairpin ribozyme-mRNA complex

6. Figure 6.11

7. Figure 6.12 DNAzymes can also be designed to cleave a target mRNA, such as the one shown here.

8. Figure 6.13 RNAi. Silencing of the expression of a specific gene by either siRNA (small interfering RNA) or shRNA (short hairpin RNA). Dicer cleaves shRNAs, while RISC (RNA-Induced Silencing Complex) cleaves the target mRNA.

9. RNA Interference (RNAi)
In 2006, Fire and Mello received a Nobel Prize for their RNAi work uisng Double Stranded RNA in C. elegans – see RNA Interference on YouTube:
Discovered in petunia - see RNAi Discovered on YouTube:

10. Figure 6.16 A. Zinc finger nucleases can be genetically engineered to specifically cut a target gene. B. A genetic construct of a zinc finger nuclease gene in E. coli under the control of the T7 promoter.

11. Genome Editing with CRISPR-Cas9 This is Huge. https://www. youtube
CRISPR-Cas9 technology allows one to edit genome sequences (delete genes, add genes, change nucleotides)
Clinical trials are just beginning in the US
see
Would you change the human germ line and eliminate deleterious disease genes or design your own child?

12. Figure 6.17 CRISPR–Cas9 represents perhaps the simplest and best way to edit (i.e., cut or cut and replace) a gene. It relies on a specific guide RNA (sgRNA) and a CAS9 gene encoding a double stranded DNA nuclease that cuts the targeted gene.

13. Figure 6. 18 Nanozymes are nanoparticles attached to specific enzymes
Figure 6.18 Nanozymes are nanoparticles attached to specific enzymes. In this case, the nanozyme is a gold nanoparticle (yellow) attached to RNase A (blue) and to specific DNA oligonucleotides (red). The nanozyme is designed to cut specific mRNAs (which are complementary to the DNA oligonucleotide sequence.
Note also that nanoparticles can also be targeted to particular cells by modifying their surface with cell-targeting aptamers.

14. Figure 6.21 Virus vectors, such as this lentivirus, are being used to deliver various therapeutic molecules (e.g., “good” genes, CRISPR-Cas9, siRNA, shRNA, etc.).

15. Virus vectors used to deliver genes for (Human) Gene Therapy
Retroviruses
Gamma-retrovirus
Lentivirus [e.g., Human immunodeficiency virus type I or HIV-1])
Adenoviruses
Adeno-associated viruses (AAV)
Herpes simplex virus (HSV-1)

16. Nonviral systems used to deliver genes for (Human) Gene Therapy
Direct injection (DNA or RNA)
Lipids (e.g., attach siRNA to cholesterol or long-chain fatty acids)
Non-pathogenic bacteria normally found in association with mammalian tissues and cells
Dendrimers-small nonimmunogenic, water-soluble nanoparticles 1-15 nm in size
Monoclonal antibodies
Aptamers
Transposons

17. Figure 6.26 Germline correction of muscular dystrophy in mice by direct injection of a CRISPR-Cas9 gene construction.

18. Human Gene Therapy (disease targets)
AIDS
Amyotrophic lateral sclerosis
Cancer
Cardiovasc. disease
Cystic fibrosis
Familial hypercholesterolemia
Gaucher disease
Hemophilia A
Hemophilia B
Hunters disease
Multiple sclerosis
Muscular dystrophy
Rheumatoid arthritis
Severe combined immunodeficiency

19. Two types of gene therapy
Ex vivo -cells are removed from the body, the gene of interest is inserted into them, the cells are cultured to increase cell numbers, and they are returned to the body by infusion or transplantation (time consuming and expensive)
In vivo -a gene is introduced directly into specific cells within the body (quick and inexpensive), but targeting certain cells (e.g., bone marrow stem cells) is difficult

20. Severe Combined ImmunoDeficiency (SCID)
See How is ADA deficiency treated?
There are no real cures for ADA deficiency, but doctors have tried to restore ADA levels and improve immune system function with a variety of treatments:
Bone marrow transplantation from a biological match (for example, a sibling) to provide healthy immune cells
Transfusions of red blood cells (containing high levels of ADA) from a healthy donor
Enzyme replacement therapy, involving repeated injections of the ADA enzyme
Gene therapy - to insert synthetic DNA containing a normal ADA gene into immune cells
6-yr-old Ashanthi DeSilva-SCID sufferer treated with gene therapy-coloring at home in N Olmstead, OH (March 1993).

21. Figure 6. 35 Overview of prodrug activation therapy
Figure 6.35 Overview of prodrug activation therapy. Tumor cells are transfected or transduced with a gene (red color) that encodes an enzyme (E) which converts an inactive precursor drug or prodrug (X) into a cytotoxic drug (X*) which kills the tumor cells and adjacent cells (bystander effect).

22. Figure 6.36 Promoterless Gene Targeting can be used to reduce unwanted activation of nearby genes, including oncogenes. A. Adverse effects can occur with promoter-target gene construct. B. No adverse effects should occur in a promoterless target gene construct.

23. Is gene therapy safe? What do you think? Jesse Gelsinger story
Jesse Gelsinger (June 18, September 17, 1999) was the first person publicly identified as having died in a clinical trial for gene therapy. He was 18 years old. Gelsinger suffered from ornithine transcarbamylase deficiency, an X-linked genetic disease of the liver, whose victims are unable to metabolize ammonia - a byproduct of protein breakdown. The disease is usually fatal at birth, but Gelsinger had not inherited the disease; in his case it was the result of a genetic mutation and as such was not as severe - some of his cells were normal which enabled him to survive on a restricted diet and special medications.
Gelsinger joined a clinical trial run by the University of Pennsylvania that aimed to correct the mutation. On Monday, September , Gelsinger was injected with adenoviruses carrying a corrected gene in the hope that it would manufacture the needed enzyme. He died four days later, apparently having suffered a massive immune response triggered by the use of the viral vector used to transport the gene into his cells. This led to multiple organ failure and brain death. Gelsinger died on Friday, September 17th at 2:30 PM.
A Food and Drug Administration (FDA) investigation concluded that the scientists involved in the trial, including the lead researcher Dr. James M. Wilson (U Penn), broke several rules of conduct:
Inclusion of Gelsinger as a substitute for another volunteer who dropped out, despite having high ammonia levels that should have led to his exclusion from the trial
Failure by the university to report that two patients had experienced serious side effects from the gene therapy
Failure to mention the deaths of monkeys given a similar treatment in the informed consent documentation.
The University of Pennsylvania later issued a rebuttal [1], but paid the parents an undisclosed amount in settlement. The Gelsinger case was a severe setback for scientists working in the field.