Effective treatment of the central nervous system (CNS) through gene therapy is a promising approach for the many genetic diseases with neurological manifestations. It’s a particular hurdle for diseases with both CNS and systemic involvement, where an effective therapy would need to work on both sides of the blood–brain barrier. Prominent among this group are lysosomal disorders (LDs), a family of monogenetic degenerative diseases which we focus on at AVROBIO, some of which have severe neurodegenerative aspects^[1].

Effectively arresting or perhaps even reversing this degeneration could spare parents the pain of watching their child fail to progress or even regress through milestones – as seen in young boys with Hunter syndrome^[2], for example. It could prevent many adults from developing genetically linked dementias such as GBA-Parkinson’s disease – seen disproportionately in people with Gaucher disease^[3]. The medical need is inarguable and is driving intense research. My colleague Geoff MacKay recently reviewed the breadth of efforts to treat CNS symptoms he saw at 2020 WORLDSymposium™, the leading LD conference^[4].

At AVROBIO, we are pursuing lentiviral gene therapy, an approach whereby hematopoietic stem cells (HSCs) are genetically modified. Lentiviral gene therapy is unique in its potential to impact symptoms from head to toe via all the nucleated components of blood, as well as the microglia in the CNS. Microglia are multi-functional and widespread throughout neuronal tissue and offer a compelling avenue to investigate the potential for their own functional correction and/or therapeutic enzyme delivery after lentiviral transduction of HSCs. Others in the industry also use this modality; across the field, transformative efficacy from a one-time treatment with lentiviral gene therapy has been demonstrated across a growing list of indications and clinical trials^[5].

Our investigational gene therapies are designed to combine optimized lentiviral vectors and personalized busulfan conditioning deploying state-of-the-art precision dosing. Busulfan is an established conditioning agent in the lentiviral gene therapy field^[6] – and it is key to the “head-to-toe” benefits we hope to demonstrate in our therapies.

Indeed, after more than 70 years of clinical use, principally in blood cancers, human data now show that busulfan is finding a new career – as a shepherd to the CNS for the descendants of transplanted HSCs, in particular the monocytes that become microglia cells^[6] (See Box 1). We are leveraging this ‘shepherding’ facility of busulfan in our own clinical programs and hope to expand in the future through continued innovation around this remarkable drug, as I recap below (Box 1).

|Box 1| Defining microglia: A primary immune cell within the CNS, and a type of macrophage. Neurologists tend to only use the term ‘microglia’ when the cell in question is derived from primitive macrophages in the yolk sac, rather than from an HSC in the bone marrow. For most intents and purposes, however, referring to them as resident macrophages, microglia-like cells or microglia is interchangeable in relation to their overall phenotype and function.

From team player to solo operator: a new role for busulfan showcases special talents

Busulfan is a clinically validated conditioning agent that has been administered as a single agent in a single cycle to hundreds of patients treated with investigational lentiviral gene therapies^[7]. A therapeutic alkylating agent, busulfan has conventionally been used as part of a cocktail of drugs for treating blood cancers.

But emerging data support busulfan as a potentially powerful tool for brain conditioning. This novel feature is due to the low level of busulfan binding to proteins in the blood; as a small molecule unencumbered by larger proteins, it can readily cross the blood–brain barrier^[8].

We believe that long-term engraftment of HSC-derived “daughter” cells following gene therapy may lead to the reconstitution of a microglial network, where the new gene-modified microglia have the potential to be functionally corrected and/or drive cross-correction of neuronal tissue via protein secretion and uptake. The key questions leading up to such a potentially outcome: Does busulfan enable genetically modified microglia to engraft in the CNS? And once there, do the microglia produce a functional enzyme? There are now early human data providing insight into these questions, drawing from clinical trials in the devastating disease metachromatic leukodystrophy (MLD), which has led the way in this area in many respects^[6],[5].

An MLD clinical story confirms a role for busulfan in addressing
CNS disease

A recent study of two patients who had received allogeneic hematopoietic stem cell (HSC) transplants for MLD with the use of busulfan showed donor-derived macrophages distributed throughout the entire white matter^[7]. Although there was no evidence of enzyme cross-correction (which would likely require the amplification of expression to supraphysiological levels), the transplanted patients had local tissue changes that suggested the donor-derived cells were making a therapeutic impact. This is encouraging histopathological evidence underpinning HSC transplantation for addressing CNS pathophysiology, and raises the exciting question of what further benefits will be achieved with the presence of gene-modified macrophages.

Happily, over the last ten years, there has been supporting evidence on that front, too. For example, in a clinical study of lentiviral gene therapy-treated patients with MLD employing busulfan conditioning, researchers observed durable, raised levels of active enzyme in the cerebrospinal fluid of treated patients, as reported by Alessandra Biffi et al^[5]. The levels and activity of the enzyme reported in the study were comparable to healthy individuals and were completely absent prior to treatment. A reduction in the toxic substrate in peripheral nerve samples was also seen. Overall, pre-symptomatic treatment was associated with protection against disease and resultant comparability on motor skills to normally developing children: the best possible result. This program is now being advanced by Orchard Therapeutics.

In summary, these case studies tell a compelling story of busulfan-supported lentiviral gene therapy making a difference in the CNS of patients. And these human studies are of course additive to many more years of supportive preclinical work. In our own Pompe disease preclinical program, we recently demonstrated in a mouse model the ability of transduced hematopoietic stem cell transplants, supported by busulfan conditioning, to reduce levels of glycogen (the pathological substrate that accumulates) to wild-type mouse levels in the brain^[9].

How does this migration across the blood–brain barrier occur? We don’t yet know. Perhaps busulfan temporarily disrupts the barrier^[10]. Perhaps monocytes cross on their own to some extent. What we do know is that in other than in very young children, adeno-associated viral (AAV) vector gene therapies, administered intravenously, do not efficiently cross the blood–brain barrier. And that large macromolecules, such as enzyme replacement therapy (ERT), also do not. ERT is the standard of care for many lysosomal disorders, but the blood–brain barrier prevents its migration through the blood–brain barrier, so the CNS symptoms develop unchallenged.

New opportunities abound

It is clear conditioning is a powerful tool for supporting lentiviral gene therapy, potentially opening the door to addressing a wide range of diseases with CNS involvement. Over the years, busulfan has benefited from an ever-evolving pool of clinician experience, and numerous improvements, including: patient education; an intravenous option; new capsule dosing to reduce pill burden; protocols for at-home and out-patient dosing (which has been reported to be associated with equivalent or superior outcomes compared to inpatient care); and more effective management, including advanced antiemetics.

It’s important to note that even with this personalized medicine approach, in our clinical trials to date we have observed anticipated side effects from busulfan, including nausea, mucositis, fever, rash and hair thinning/loss, which typically came on quickly with a peak of three to five days and resolved quickly. Busulfan, indicated to be dosed in combination with cyclophosphamide to treat chronic myeloid leukemia, may cause temporary or permanent infertility when injected prior to allogeneic (or donor) HSC transplantation. In our lentiviral gene therapy trials, we use personalized busulfan as a single agent for a single cycle followed by infusion of the patient’s own genetically modified HSCs cells, and the potential risk of infertility due to busulfan in this setting is still being studied.

And there’s even more we can do to advance the potential of this conditioning agent. Specifically, precision around cumulative tissue exposure is crucial, with a target range of 78–101 mg.hr/L to maximize the potential for long-term engraftment while avoiding toxicities associated with out-of-range exposure^[11]. Our target is a cumulative AUC of 90 mg.hr/L, a regimen we refer to in shorthand as “Bu90.” Achieving this target range is a challenge because busulfan is metabolized differently from patient to patient, and even from day to day within the same patient. AVROBIO is leading the way by not just aiming to be in range but targeting the actual midpoint in order to further enhance safety and efficacy. We deploy a personalized medicine approach involving daily blood monitoring to assess the rate of busulfan metabolism and precisely adjust the next day’s dose accordingly in order to hit our cumulative target of 90 mg.hr/L. The challenge with busulfan is the high variable relationship between the dose and resulting blood concentration. The degree of intra- and inter-patient pharmacokinetic variability can be as much as 10x.

This type of precision targeting of the cumulative exposure resulting from a single agent, which takes place over four days, differs from the ‘traditional’ use of alkylating agents as part of potent drug cocktails aimed at eliminating cancer cells, with the risks associated with polypharmacy. A personalized medicine approach aims to achieve a more consistent targeted exposure through a combination of readily available point-of-care busulfan plasma concentration measurement and targeting the mid-point of the range. This method significantly refines traditional therapeutic drug monitoring (TDM), which typically targets a range, hence there is the potential if at the outer edges of the range to increase the risk of out-of-range toxicity (upper end) or reduced engraftment (lower end).

We recently undertook a collaboration with the aim of enabling the widespread deployment of precision targeting for cumulative busulfan exposure through a collaboration with Saladax Biomedical^[12]. This collaboration is intended to deliver a novel nanoparticle-immunoassay kit compatible with automated analytical devices commonly used at hospitals and clinics. It is designed to enable much faster, on-site analysis of busulfan metabolism – minutes as opposed to hours. We hope this will greatly expand access to personalized busulfan conditioning deploying state-of-the-art precision dosing, for use not just in the field of lentiviral gene therapy, but across a wide range of HSC transplants for conditions ranging from hematological cancers to autoimmune disease.

It has been incredibly exciting to see the emergence of a technology that can work both sides of the blood–brain barrier and potentially halt, or potentially in some instances reverse, CNS and systemic changes across a range of devastating diseases. Busulfan’s role in that process, after decades of unrelated use in hematological oncology, is a reminder that established drugs can often have new uses, and that biology is a strange and wonderful thing. Ultimately, there’s increased potential for busulfan to serve as a shepherd of gene-modified cells to impact CNS diseases, and AVROBIO is excited to have the opportunity to be a leader in this next frontier for lentiviral gene therapy.

Originally published by Cell & Gene

Chris Mason, M.D., Ph.D. is the Chief Scientific Officer of AVROBIO, Inc. 
AVROBIO is currently conducting clinical trials to evaluate the safety and efficacy of its investigational lentiviral gene therapies. None of these investigational gene therapies has been approved by the U.S. Food and Drug Administration or any other regulatory agency. For more information, go to www.avrobio.com.