Timothy Ray Brown is an anomaly.

Now known worldwide as The Berlin Patient, in

1995, he was merely a university student in Berlin who tested positive for human

immunodeficiency virus (HIV). He spent the next ten years on antiretroviral therapy (ART), a

combination of HIV medicines taken every day that reduces the amount of HIV in ones body.

But Brown also developed acute myeloid leukemia, so his oncologist decided on a stem cell

transplant (Cohen, 2014). Stem cell transplants are notorious for the difficulty in finding a match

between donor and transplant, and Browns potential transplant had another condition: finding a

donor homozygous for a rare CCR5 gene mutation that provides natural resistance to HIV. The

61st match tested, however, did possess the CCR5 mutation, and Brown began conditioning,

destroying his immune system with chemotherapy and radiation treatment so it could be replaced

with transplanted immune cells (Engel, 2015). On February 7, 2007, Brown received the stem

cell transplant. He stopped antiretroviral therapy on the same day. Typically, when HIV-infected

people stop taking antiretroviral drugs, their HIV levels become extraordinarily high in weeks

(Cohen, 2014). Nine years later, though, Brown remains HIV-free, the only human cured of HIV.

Browns recovery seems nearly miraculous when faced with the grave toll that HIV has

inflicted globally. HIV is a retrovirus that causes acquired immunodeficiency disease (AIDS) by

attacking the CD4 T-cells; this serious sabotage of the immune system facilitates infections and

cancers that the body is subsequently too weak to fight off. AIDS is considered a late stage of

HIV infection, where CD4 cell count has declined to below 200 cells per cubic millimeter of

blood, as opposed to a healthy range of 500 to 1600 cells/mm3 (HIV/AIDS Basics, 2016). HIV

is transmitted through certain bodily fluids blood, semen, vaginal fluids, and breast milk

usually as the result of unprotected needle use or sexual activity. There are currently 36.7 million

people, including 1.8 million children, worldwide living with HIV, with the majority of infected
individuals in low-income regions such as sub-Saharan Africa (HIV/AIDS Basics, 2016). The

sheer magnitude of the HIV epidemic is devastating and growing daily. Yet existing treatments

today are regrettably temporary. Treatments such as highly active antiretroviral therapy

(HAART) may prolong lives but fail to eradicate HIV from the body and may even contribute to

cardiovascular disorders (Peterson et al., 2013). Other disadvantages of HAART include its high

cost and possibility of burgeoning drug resistance (Manjunath et al., 2013). This lack of a

comprehensive cure necessitates developing gene therapy approaches to treating HIV, one of

which involves disruption of the CCR5 coreceptor loci in CD4 cells. Researchers have been

inspired to pursue this method by the rare homozygous CCR5 delta-32 deletion, used in the

Berlin Patients stem cell transplant, which stops CD4 cells from developing a CCR5 receptor,

thus preventing HIV from entering the cell (Engel, 2015). People heterozygous for CCR5 delta-

32 with HIV have also been noted to experience a slower progression towards AIDS (Tebas et

al., 2014). Overall, CCR5 is an ideal target for disruption because CCR5 inhibition does not

affect immune expression detrimentally while conferring the vital advantage of HIV resistance

(Peterson et al., 2013).

Gene therapy tools used to alter CCR5 synthesis include zinc finger nucleases (ZFNs)

and transcription activator-like effector nucleases (TALENs). These engineered nucleases disrupt

CCR5 by recognizing certain DNA sequences and then cleaving DNA double-strand breaks

(DSBs) at those loci. Then, random insertions or deletions modify the cleavage site through a

nonhomologous end joining (NHEJ) repair pathway, or larger gene fragments are added through

homology-directed repair (HDR) (Wang & Cannon, 2016). In one of the first pre-clinical trials,

for example, a ZFN pair created a DSB whose repair required a 5 base pair duplication. This

addition shifted the reading frame and introduced two premature stop codons, thus inhibiting
CCR5 expression. 33% of the modified cells had CCR5 disrupted on both alleles (Wang &

Cannon, 2016). Trials on human patients are still underway. The patients in the Sangamo trial,

which utilized ZFNs for CCR5 disruptions, all rebounded in HIV levels after ART was

discontinued, although one patient heterozygous for the CCR5 delta-32 deletion gained control

of the viral replication without ART (Htter et al., 2015). Researchers are now investigating the

potential pairing of CCR5 disruption with the delivery and integration of chimeric antigen

receptors (CAR) into the genome (Ibarra et al., 2016). Galvanized by the success of the Berlin

Patient, they have turned to gene therapy in search of a permanent HIV cure.
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March 10, 2017, from Science website: http://www.sciencemag.org/news/2014/09/how-

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Engel, M. (2015, February 27). Doctor who cured Berlin patient of HIV: We knew we were

doing something very special. Retrieved March 10, 2017, from Fred Hutch website:

http://www.fredhutch.org/en/news/center-news/2015/02/timothy-ray-brown-doctor-who-

cured-him.html

[HIV/AIDS basics]. (2016). Retrieved March 10, 2017, from AIDS.gov website:

https://www.aids.gov/hiv-aids-basics/index.html

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