From the Editors: Cell and Gene Therapy: A Potential Functional Cure for Human Immunodeficiency Virus Infection

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Shyam Bhakta, MD, MBA
National Center for Regenerative Medicine
Case Western Reserve University
Cleveland, Ohio, USA

Human immunodeficiency virus (HIV) remains a significant global challenge and demands continuous progress in therapeutic approaches (1). Since the start of the epidemic, 85 million people have been infected with HIV, and 40 million persons have died (2). However, over the ensuing 35 years, HIV has evolved from a uniformly fatal disease to one that is a serious albeit manageable lifelong illness (2). According to the World Health Organization in 2022, 1.3 million people were infected with HIV and 630,000 persons died from HIV-related causes. Over 63,000 new cases were diagnosed in the seven major markets (7MM), which consist of the United States, United Kingdom, Germany, France, Italy, Spain, and Japan (3). Currently, there are 39 million people globally living with HIV (4).

During primary HIV infection, although a robust virus-specific CD4-mediated cytotoxic T-lymphocyte (CTL) response is associated with initial control of viremia, diversification of the virus accompanied by limited functionality of the CTL response renders the immune system unable to clear the virus. In addition, HIV-specific CD4 T-cells critical for maintaining an effective and sustained anti-HIV immune response are also preferential targets for HIV infection. Integration of the retroviral genome into the host cell DNA results in continuous infection and eventual loss of CD4 T-cells. Declining T-cells accompanied by viremia leads to T-cell depletion and failure to generate an effective memory response. This process, if left untreated, progresses to acquired immune deficiency syndrome (AIDS) (1).

Chronic HIV infection destroys CD4 T-cell immunity, leading to immune system dysfunction and inability to control HIV (5). Studies of HIV-positive patients demonstrated the importance of CD4 T-cells specific for HIV in controlling viremia and reducing disease progression. Gag-specific CD4 T cells during acute HIV infection are associated with improved viral control. In studies of blood transfusion-acquired HIV-1 infection, robust and sustained CD4 T-cell proliferation responses to the Gag p24 antigen correlated with viremia control and lack of disease progression (5). Depletion of Gag-specific CD4+ T-cells and circulating levels in peripheral blood are correlated negatively with viremia.

Highly active antiretroviral therapy (HAART) was developed in the early 1990s (2). While HAART allows for durable, long-term remission of HIV infection, downsides include the need for lifelong therapy, strict adherence, long-term toxicities, potential development of antiretroviral drug resistance, and inability to eradicate latent virus. Although HAART restores CD4 counts, HIV-specific CD4 T-cells are not restored fully. Upon cessation of antiretroviral therapy, only a modest CTL response is generated and it is ineffective in mediating viral suppression or clearance (1).

In the United States, 45,000 people with HIV fail first-line antiretroviral therapy, and 91,000 patients are nonadherent, contributing to 35,000 - 40,000 new infections each year. Patients who fail first-line treatment are more likely to fail again with second-line medications as well as experience side effects, treatment fatigue, and drug resistance. Drug resistance-associated viral mutations continue to pose a threat because, once mutations are transmitted, it may be harder to treat new patients with current medications. Given the long history of stigma and discrimination against persons living with HIV, this can lead to inconsistent timing of or altogether forgetting to take medication. Finally, patients with HIV have an increased risk of depression and substance abuse, both of which increase nonadherence (2).

Experimental therapies include therapeutic vaccines, genetically modified immune cells that are resistant to HIV infection, cure-inducing immunotherapies, interventions to permanently silence HIV-infected cells, and drugs that reactivate latent viral reservoirs that can be eradicated by cytotoxic T-lymphocytes (1). Unfortunately, in January 2023, a phase 3 vaccine trial sponsored by Johnson and Johnson was terminated due to lack of efficacy, delivering another big blow to the HIV landscape in terms of a therapeutic vaccine (3).

Adoptive, autologous and antigen-specific CD4 T-cell therapy is a promising strategy to reconstitute the immune system against HIV that may help improve clinical outcomes and reduce dependence on antiretroviral therapy. Although HIV-specific CD4 T-cells are found in the peripheral blood in almost all patients with HIV, their percentages are very low. HIV-specific CD4 T-cells that are able to avoid depletion demonstrate functional impairment. Previous protocols for expanding tumor antigen-specific T-cells were utilized for expanding HIV-specific T-cells but were unsuccessful (5). CD4 T-cell modification for HIV resistance may explain these cells’ ability to persist in vivo. Clinical trials including those for chimeric antigen receptor T-cell (CAR-T) therapy have demonstrated safety of lentivirus-modified cell infusion (5). According to GlobalData, in 2023, the HIV therapeutics market in the 7MM was valued at $27 billion (3). In the United States, the current average lifetime cost of HIV treatment is $1.7 million per person (4).

Strategies to protect the CD4 T-cells from new infections that can elicit a sustained CTL response in the presence of viremia are vital for a functional cure against HIV. In addition to insufficient levels of HIV-specific CD4 helper cells in HIV patients, another major obstacle to viral clearance is the presence of latent viral reservoir in immune cells. Due to limited transcription of the integrated provirus, latently infected cells cannot be targeted by immune effector mechanisms and present themselves as a source of viremia that can result in further depletion of the CD4 cells (1).

Multiple cell and gene therapy products for HIV therapy have been developed. Bacterial MazF endoribonuclease, when infused as an autologous product, is expressed in CD4+ cells and has higher activity against HIV RNA compared to endogenous host RNA. However, following interruption of antiretroviral therapy, all patients experience rebound viremia. Similar results were seen in trials with antisense RNA and zinc-finger endonuclease deletion of CCR5 genes. While cell and gene therapies for HIV are safe and well-tolerated, thus far, none have succeeded in achieving durable control over viral replication (6). Other strategies devised to protect infused CD4 T-cells from HIV include overexpression of restriction factors, use of fusion inhibitors, targeting HIV coreceptors through RNA and gene editing techniques, targeting HIV genes through small interfering RNA (siRNA), and dominant negative genes (1). Efforts in the field of immune, gene, and cell therapies have provided encouraging data that suggests that enhancing the immune response of patients while suppressing HIV genes has a promising shot towards eradicating HIV (1).

During CD4 cell infection, HIV binds to one of two CD4 cell co-receptors, CCR5 and CXCR4. CCR5-tropic strains predominate during early infection, whereas CXCR4-tropic strains arise during disease progression (1). Rare individuals, known as natural or elite controllers, who maintain low levels of HIV viremia without antiretroviral therapy, demonstrate high levels of Gag-specific CD4+ T cells. Such individuals may also have lower levels of CCR5 cell surface receptor expression (6). HIV remission in the “Berlin patient” was attributed to a natural Δ32 homozygous mutation in the CCR5 gene that resulted in a non-functional co-receptor (1).

Li et al. published their work detailing the preclinical development and clinical-scale manufacturing of HIV Gag-specific, lentivirus-modified CD4 cells for an HIV functional cure (5). AGT103 (American Gene Technologies [AGT], Rockville, MD; https://www.americangene.com) is an inhibitory RNA recombinant lentiviral vector targeting the chemokine receptor type 5 (CCR5) coreceptor, necessary for CD4 T-cell infection, and the viral infectivity factor/transactivator of transcription (VIF/TAT) HIV coding sequences necessary for virus assembly.

AGT103 expresses three inhibitory microRNAs (miRNAs) within a single transcript driven by the RNA polymerase II promoter elongation factor 1-alpha (EF-1 alpha). Transcription generates RNA containing three miRNA hairpin structures with targeting sequences specific for: (1) CCR5, (2) HIV TAT, and (3) HIV VIF genes. A CCR5 targeting sequence is embedded within the naturally occurring human miR30, a HIV TAT targeting sequence is embedded within miR185, and a HIV VIF targeting sequence is embedded within miR21. The RNA transcript is processed by cellular factors to produce active miRNA.

To demonstrate the effects of AGT103 on CCR5 expression, JC53 cells, which are HeLa cells that express high levels of CD4 and CCR5, were transduced with either lentivirus control or AGT103. Flow cytometry demonstrated that AGT103 reduced CCR5 expression by over 98 percent. The specificity of AGT103 was tested by transducing Vϒ9Vδ2 cells, which express both CCR2 and CCR5. While the CCR2 and CCR5 coding regions are 75 percent homologous, CCR5-specific miRNA does not target sequences shared between these two cell surface markers. AGT103 significantly reduced CCR5 expression without affecting CCR2 expression. To test for effects on common cell-surface proteins, the CEM.NKR.CCR5 cell line was used to demonstrate that AGT103 specifically decreased CCR5 expression without affecting expression of other cell-surface molecules.

JC53 cells were transduced with AGT103-GFP followed by infection with R5-tropic BaL HIV pseudovirus. Compared to control lentivirus, AGT103 reduced HIV infection of cells by over 90 percent. J1.1 HIV-latently infected cells were cloned and transduced by miRNA against the VIF and TAT sequences, which resulted in significantly reduced HIV release following induction by tumor necrosis factor-alpha (TNF-alpha). AGT103 transduction of human CD4 cells protected against HIV replication in a dose-dependent manner. Finally, AGT103 transduction of human CD4 T-cells resulted in dose-dependent protection against HIV infection.

The authors developed a protocol for expanding HIV Gag-specific CD4 T-cells that consisted of expanding peripheral blood mononuclear cells (PBMCs) from HIV-positive individuals by stimulation with HIV Gag protein and other peptides followed by stimulation with CD3/CD28 Dynabeads and transduction with lentivirus, resulting in increased expansion of Gag-specific CD3 T-cells that was improved further by depletion of non-CD4 cell types, including CD8 T-cells. AGT103 targets CCR5 and HIV TAT/VIF and is effective at inhibiting HIV infection, replication, and virus release from latently-infected cells.

Muvarak et al. conducted a phase 1 trial to study the safety and durability of AGT103-T autologous T cell therapy for HIV infection (6). A total of 13 patients were enrolled. Patient median age was 41 (range = 26 - 59) years old. Median CD4+ T-cell count was 577/uL. Median duration of HIV infection was 14.2 (range 3.8 - 28.4) years. Median duration of antiretroviral therapy was six (range = 3 - 24) years. A total of seven patients were treated with cyclophosphamide (1 g/m2) one week prior to receiving either low (< 109) or high (> 109) genetically modified CD4+ T cells, delivering between 2 and 21 million cells/kg body weight. Following infusion, transgene copy number was measured by quantitative polymerase chain reaction (qPCR).

Cell therapy was safe and demonstrated only mild adverse effects without any serious adverse effects. AGT103 transgene was detected in six of seven patients up to six months following infusion. Cells modified by AGT103 were detected in most participants at six months post-infusion and peaked at three days following infusion. At 14 days following infusion, PBMCs were analyzed for Gag-specific CD4+ T-cells, which were increased 9- 300-fold from baseline, with levels remaining 2-70-fold higher up to 6 months following infusion. The authors suggest that Gag-specific CD4+ T-cells will assist B-cells to improve production of potent neutralizing antibodies and support differentiation of CD8+ cytotoxic T-lymphocytes capable of recognizing and suppressing viral escape variants.

Jain et al. studied multiple antiretroviral therapy treatment interruptions in patients following treatment with HIV-specific CD4 T-cells (1). The authors demonstrated that, following the initial therapy interruption, all patients demonstrated an expected rise in viremia that was accompanied by an increase in HIV-specific CD8 T-cells. Following a second interruption of antiretroviral therapy, Gag-specific CD8 T-cell levels were either maintained or increased and that peak viremia decreased. The authors also demonstrated that, following resumption of antiretroviral therapy, viremia decreased more rapidly without drug resistance or other adverse events. The authors conclude that AGT103-T may contribute to a resetting of the immune response to that generated during primary infection (1).

In August 2023, American Gene Technologies announced that Addimmune, a subsidiary spinoff, will merge with the special purpose acquisition company 10X Capital Venture Acquisition Corp, which is also a minority investor in American Gene Technologies. Addimmune will launch a phase 1b trial to investigate AGT103-T to review its safety and efficacy as monotherapy in patients who will not receive standard treatment. This trial will enroll 24 patients who will similarly undergo leukapharesis to remove their T-cells followed by dosing with AGT103-T and re-introduction into the body. Patients will be randomized into one of three study arms. In the first arm, patients will be taken off of antiretroviral therapy immediately. The second arm of patients will receive treatment interruption after 14 days, and the third arm of patients will receive treatment interruption after 28 days. All patients will be followed for 16 weeks (3).

The gene and cell therapy market may be valued at $5 trillion, almost four times the size of the current pharmaceutical market. Improved therapeutics with more convenience and lower toxicities that offer a one-and-done functional cure is a substantial market waiting to be tapped (4).

Disclosure: Dr. Bhakta is a member of the Alliance for Regenerative Medicine and a shareholder in American Gene Technologies, Inc. and Biobuzz Networks, Inc.

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