Gene therapy for HIV/AIDS Flashcards
How many people (globally) HIV-infected? How many of those are not on treatment?
39 million, of which 9.2 million not on treatment
What is the number of yearly AIDS-related deaths?
630.000
What is the number of new HIV-infections/year
1.3 million
What is cART?
Combination antiretroviral therapy -> a combination of 2 or more antiretroviral drugs used to suppress HIV and prevent resistance
What are the downsides of cART for HIV?
- Life-long medication -> cannot cure HIV
- Various long-term side effects
- Interaction with other substances (medication, supplements)
- Resistance of the virus
How many HIV-infected individuals are there in The Netherlands? What is the biggest group?
~22.500, biggest group = MSM
How many new HIV infections occur yearly in The Netherlands?
~250
What is the HIV infection cycle? (8)
- Binding to CD4+ & co-receptor
- Fusion & release of viral material
- RNA reverse transcribed into DNA
- Synthesis of a second DNA strand -> dsDNA
- dsDNA is translocated into the nucleus & integrated into the host genome
- HIV provirus acts as template for new RNA transcripts
- Translation of viral RNA allows for production of new viral proteins
- New viral particles are formed & released by budding of the cell membrane
What is HIV provirus?
Viral dsDNA integrated into the host genome
What is the downside of cART therapy in terms of latency?
cART does not eliminate the latent reservoir -> 1/100.000 CD4+ T-cells will have provirus integrated into the genome
True or false: HIV cure is currently impossible
False; functional HIV cure is possible through bone marrow transplantation with specific donor properties, no longer allowing HIV to infect CD4+ T-cells
What is the difference between eradicating cure & functional cure?
Eredication = all replication-competent HIV provirus completely removed from the body
Funtional cure = no viral load detectable, no need for cART
Why is bone marrow transplantation to get rid of HIV not routinely performed?
Very risky procedure, high risk of mortality
Which forms of regulatory RNA are there?
- siRNA = small interfering RNA
- miRNA = microRNA
What is the origin of siRNA?
Derived from longer dsRNA
What is the origin of miRNA?
Derived from RNA transcripts of specific miRNA genes
What is the average length of miRNA?
20-25 nucleotides
How many human miRNAs are known?
> 1000
What are the functions of miRNA and siRNA?
Regulation of gene transcription
How are miRNA & siRNA processed & used for regulation of transcription? (3)
- Cut into shorter fragments by DICER
- Bound to Argonout-2, which (together with other proteins) forms the RISC-complex
- RISC-complex uses miRNA & siRNA to detect complementary strands, after which their transcriptional activity is suppressed, or they are degraded
How can ectopic RNAi elements for therapeutic purposed be introduced into cells? (2)
- Viral vectors to encode for short hairpin RNAs
- Direct introduction of siRNA (e.g. electroporation)
What is the advantage of RNAi gene therapy over CRISPR-Cas?
All proteins for RNAi are already present in cells -> no need to introduce foreign peptides like in CRISPR-Cas
What is a possible candidate gene for silencing, leading to less HIV viral load?
CCR5 or CXCR4 = HIV co-receptors
What are the effects of RNAi to suppress CCR5 as a gene therapeutic approach for HIV? (3)
- Potent miRNAs can be gerneated against CCR5 -> effective reduction of CCR5 expression
- Sustained antiviral effects
- Absence of cytotoxic effects
What is the disadvantage of RNAi targeting a single gene to reduce HIV viral load? What is a solution to this?
Virus can mutate to no longer rely on the suppressed gene
Solution: target multiple anti-HIV genes to avoid escape
Why is it target to target viral RNAs with RNAi?
It is often covered with protein -> not accessible to RNAi complex
What is the downside for RNAi with regards to HIV latency? How can this be solved?
The life-span of miRNA for RNAi is limited -> needs renewed dosing to keep HIV suppressed
Solution: using a lentiviral vector to integrate anti-HIV miRNA genes into the host genome
Which HIV-processes can be targeted using CRISPR-Cas?
- Degradation of viral dsDNA outside the nucleus
- DNA repair of DNA provirus integrated into the host genome
- Degradation of viral RNA/mRNA
What is the original purpose of CRISPR-Cas?
Bacterial defence mechanism against viral infections
How is CRISPR-Cas targeted?
Using a targetRNA that guides Cas endonucleases to the target gene
What is the difference between different Cas molecules?
They all have different targets & ways of cutting genetic material
What are the properties of Cas9? (3)
- Targets dsDNA
- Leaves blunt ends
- Requires PAM recognition sequence to bind to DNA
What are the properties of Cas12? (3)
- Targets dsDNA
- Leaves staggered ends
- Requires PAM recognition sequence to bind to DNA
What are the properties of Cas13? (2)
- Targets ssRNA
- Does not require PAM recognition sequence
What is the difficulty with Cas-molecules that require PAM recognition to bind to DNA?
It is not always possible to find a suitable PAM in the vicinity of the target sequence
What is the result of CRISPR-Cas cutting of DNA?
Initiation of repair mechanisms
By which ways can CRISPR-Cas-induced DNA damage be repaired? (2) What are their effects?
- Non-homologous end-joining (NHEJ) -> insertions/deletions in gene, causing it to be disabled
- Homology-directed repair (HDR) -> leads to exact repair based on the complementary DNA template, but can also be used to insert a donor template
What are requirements of good HIV gene therapy targets? (2)
- Target sequences must be essential for HIV -> less chance of escape
- Target must be conserved in all HIV viral sequences -> allows for targeting of all strains
What are possible delivery mechanisms for HIV gene therapy? (3)
- Chemical (e.g. lipid nanoparticles)
- Viral: viral vectors
- Physical: sonoporation/electroporation
What are potential viral delivery systems for anti HIV gene therapy? (3)
- Retrovirus/lentivirus vectors
- Adenovirus vectors
- Adeno-associated viruses
What is the advantage and at the same time disadvantage of using retrovirus/lentiviral vectors for HIV gene therapy?
Leads to integration of genes into host genome -> constitutive expression
What is a problem of using adenovirus vectors or adeno-associated viruses to deliver HIV gene therapy?
Repeated administration can cause immunity to the vector
What is a challenge in trying to deliver CRISPR-Cas gene therapy?
CAS molecules can be rather large -> cannot always be packed into a vector
What are solutions to be able to deliver large CAS molecules for HIV gene therapy? (2)
- Using multiple vectors, each encoding part of the molecule
- Using smaller Cas orthologues
What is the problem of using multiple vectors to deliver parts of Cas molecules for gene therapy?
It is hard to ensure that a cell is infected by multiple vectors
Why are lentiviruses no longer used for gene therapy delivery in humans?
Safety concerns (oncogenic risk)
Where is the majority of HIV gene therapy trials located?
USA
Has HIV gene therapy been successfully applied in humans?
From a safety point of view: yes
But: no reduction of viral load
